Comparative ligand mapping from mhc class i  positive cells

ABSTRACT

The present invention relates generally to a methodology for the isolation, purification and identification of peptide ligands presented by MHC positive cells. In particular, the methodology of the present invention relates to the isolation, purification and identification of these peptide ligands from soluble class I and class II MHC molecules which may be uninfected, infected, or tumorgenic. The methodology of the present invention broadly allows for these peptide ligands and their comcomittant source proteins thereof to be identified and used as markers for infected versus uninfected cells and/or tumorgenic versus nontumorgenic cells with said identification being useful for marking or targeting a cell for therapeutic treatment or priming the immune response against infected cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 12/951,588, filed Nov. 22,2010; which is a continuation of U.S. Ser. No. 11/601,058, filed Nov.17, 2006, now abandoned; which is a divisional of U.S. Ser. No.09/974,366, filed Oct. 10, 2001, now U.S. Pat. No. 7,541,429, issuedJun. 2, 2009; which claims benefit under 35 U.S.C. 119(e) of provisionalpatent applications U.S. Ser. No. 60/240,143, filed Oct. 10, 2000; U.S.Ser. No. 60/299,452, filed Jun. 20, 2001; U.S. Ser. No. 60/256,410,filed Dec. 18, 2000; U.S. Ser. No. 60/256,409, filed Dec. 18, 2000; andU.S. Ser. No. 60/327,907, filed Oct. 9, 2001. The contents of each ofthe above-referenced patent applications are hereby expresslyincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract NumberNO1-AI-95360 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to a methodology for theisolation, purification and identification of peptide ligands presentedby MHC positive cells. In particular, the methodology of the presentinvention relates to the isolation, purification and identification ofthese peptide ligands from soluble class I and class II MHC moleculeswhich may be uninfected, infected, or tumorigenic. The methodology ofthe present invention broadly allows for these peptide ligands and theirconcomitant source proteins thereof to be identified and used as markersfor infected versus uninfected cells and/or tumorigenic versusnontumorigenic cells with said identification being useful for markingor targeting a cell for therapeutic treatment or priming the immuneresponse against infected cells.

2. Description of the Background Art

Class I major histocompatibility complex (MHC) molecules, designated HLAclass I in humans, bind and display peptide antigen ligands upon thecell surface. The peptide antigen ligands presented by the class I MHCmolecule are derived from either normal endogenous proteins (“self”) orforeign proteins (“nonself”) introduced into the cell. Nonself proteinsmay be products of malignant transformation or intracellular pathogenssuch as viruses. In this manner, class I MHC molecules conveyinformation regarding the internal fitness of a cell to immune effectorcells including but not limited to, CD8⁺ cytotoxic T lymphocytes (CTLs),which are activated upon interaction with “nonself” peptides, therebylysing or killing the cell presenting such “nonself” peptides.

Class II MHC molecules, designated HLA class II in humans, also bind anddisplay peptide antigen ligands upon the cell surface. Unlike class IMHC molecules which are expressed on virtually all nucleated cells,class II MHC molecules are normally confined to specialized cells, suchas B lymphocytes, macrophages, dendritic cells, and other antigenpresenting cells which take up foreign antigens from the extracellularfluid via an endocytic pathway. The peptides they bind and present arederived from extracellular foreign antigens, such as products ofbacteria that multiply outside of cells, wherein such products includeprotein toxins secreted by the bacteria that often times havedeleterious and even lethal effects on the host (e.g., human). In thismanner, class II molecules convey information regarding the fitness ofthe extracellular space in the vicinity of the cell displaying the classII molecule to immune effector cells, including but not limited to, CD4⁺helper T cells, thereby helping to eliminate such pathogens theexamination of such pathogens is accomplished by both helping B cellsmake antibodies against microbes, as well as toxins produced by suchmicrobes, and by activating macrophages to destroy ingested microbes.

Class I and class II HLA molecules exhibit extensive polymorphismgenerated by systematic recombinatorial and point mutation events; assuch, hundreds of different HLA types exist throughout the world'spopulation, resulting in a large immunological diversity. Such extensiveHLA diversity throughout the population results in tissue or organtransplant rejection between individuals as well as differingsusceptibilities and/or resistances to infectious diseases. HLAmolecules also contribute significantly to autoimmunity and cancer.Because HLA molecules mediate most, if not all, adaptive immuneresponses, large quantities of pure isolated HLA proteins are requiredin order to effectively study transplantation, autoimmunity disorders,and for vaccine development.

There are several applications in which purified, individual class I andclass II MHC proteins are highly useful. Such applications include usingMHC-peptide multimers as immunodiagnostic reagents for diseaseresistance/autoimmunity; assessing the binding of potentiallytherapeutic peptides; elution of peptides from MHC molecules to identifyvaccine candidates; screening transplant patients for preformed MHCspecific antibodies; and removal of anti-HLA antibodies from a patient.Since every individual has differing MHC molecules, the testing ofnumerous individual MHC molecules is a prerequisite for understandingthe differences in disease susceptibility between individuals.Therefore, purified MHC molecules representative of the hundreds ofdifferent HLA types existing throughout the world's population arehighly desirable for unraveling disease susceptibilities andresistances, as well as for designing therapeutics such as vaccines.

Class I HLA molecules alert the immune response to disorders within hostcells. Peptides, which are derived from viral- and tumor-specificproteins within the cell, are loaded into the class I molecule's antigenbinding groove in the endoplasmic reticulum of the cell and subsequentlycarried to the cell surface. Once the class I HLA molecule and itsloaded peptide ligand are on the cell surface, the class I molecule andits peptide ligand are accessible to cytotoxic T lymphocytes (CTL). CTLsurvey the peptides presented by the class I molecule and destroy thosecells harboring ligands derived from infectious or neoplastic agentswithin that cell.

While specific CTL targets have been identified, little is known aboutthe breadth and nature of ligands presented on the surface of a diseasedcell. From a basic science perspective, many outstanding questions havepermeated through the art regarding peptide exhibition. For instance, ithas been demonstrated that a virus can preferentially block expressionof HLA class I molecules from a given locus while leaving expression atother loci intact. Similarly, there are numerous reports of cancerouscells that fail to express class I HLA at particular loci. However,there are no data describing how (or if) the three classical HLA class Iloci differ in the immunoregulatory ligands they bind. It is thereforeunclear how class I molecules from the different loci vary in theirinteraction with viral- and tumor-derived ligands and the number ofpeptides each will present.

Discerning virus- and tumor-specific ligands for CTL recognition is animportant component of vaccine design. Ligands unique to tumorigenic orinfected cells can be tested and incorporated into vaccines designed toevoke a protective CTL response. Several methodologies are currentlyemployed to identify potentially protective peptide ligands. Oneapproach uses T cell lines or clones to screen for biologically activeligands among chromatographic fractions of eluted peptides. (Cox et al.,Science, vol 264, 1994, pages 716-719, which is expressly incorporatedherein by reference in its entirety) This approach has been employed toidentify peptides ligands specific to cancerous cells. A secondtechnique utilizes predictive algorithms to identify peptides capable ofbinding to a particular class I molecule based upon previouslydetermined motif and/or individual ligand sequences. (De Groot et al.,Emerging Infectious Diseases, (7) 4, 2001, which is expresslyincorporated herein by reference in its entirety) Peptides having highpredicted probability of binding from a pathogen of interest can then besynthesized and tested for T cell reactivity in precursor, tetramer orELISpot assays.

However, there has been no readily available source of individual HLAmolecules. The quantities of HLA protein available have been small andtypically consist of a mixture of different HLA molecules. Production ofHLA molecules traditionally involves growth and lysis of cellsexpressing multiple HLA molecules. Ninety percent of the population isheterozygous at each of the HLA loci; codominant expression results inmultiple HLA proteins expressed at each HLA locus. To purify nativeclass I or class II molecules from mammalian cells requirestime-consuming and cumbersome purification methods, and since each celltypically expresses multiple surface-bound HLA class I or class IImolecules, HLA purification results in a mixture of many different HLAclass I or class II molecules. When performing experiments using such amixture of HLA molecules or performing experiments using a cell havingmultiple surface-bound HLA molecules, interpretation of results cannotdirectly distinguish between the different HLA molecules, and one cannotbe certain that any particular HLA molecule is responsible for a givenresult. Therefore, a need existed in the art for a method of producingsubstantial quantities of individual HLA class I or class II moleculesso that they can be readily purified and isolated independent of otherHLA class I or class II molecules. Such individual HLA molecules, whenprovided in sufficient quantity and purity, would provide a powerfultool for studying and measuring immune responses.

Therefore, there exists a need in the art for improved methods ofepitope discovery and comparative ligand mapping for class I and classII MHC molecules, including methods of distinguishing an infected/tumorcell from an uninfected/non-tumor cell. The present invention solvesthis need by coupling the production of soluble HLA molecules with anepitope isolation, discovery, and direct comparison methodology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Overview of 2 stage PCR strategy to amplify a truncated versionof the human class I MHC.

FIG. 2. Edman sequence analysis of soluble B*1501, B*1501-HIS andB*1501-FLAG. Residue intensity was categorized as either dominant(3.5-fold or more picomolar increase over previous round) or strong (2.5to 3.5-fold increase over prior round).

FIG. 3. Representative MS ion maps from soluble B*1501, B*1501-HIS andB*1501-FLAG illustrating ion overlap between the molecules. Pooled,acid-eluted peptides were fractionated by RP-HPLC, and the individualfractions were MS scanned.

FIG. 4. Fragmentation pattern generated by MS/MS on an ion selected fromfraction 11 of B*1501, B*1501-HIS and B*1501-FLAG peptides illustratinga sequence-level overlap between the three molecules.

FIG. 5. Flow chart of the epitope discovery of C-terminal-tagged sHLAmolecules. Class I positive transfectants are infected with a pathogenof choice and sHLA preferentially purified utilizing the tag.Subtractive comparison of MS ion maps yields ions present only ininfected cell, which are then MS/MS sequenced to derive class Iepitopes.

FIG. 6. MS ion map from soluble B*0702 SupT1 cells uninfected andinfected with HIV MN-1. Pooled, acid-eluted peptides were fractionatedby RP-HPLC, and fraction #30 was MS scanned.

FIG. 7. MS ion map similar to FIG. 6 but zoomed in on the area from482-488 amu to more clearly identify all ions in the immediate area.

FIG. 8. Fragmentation pattern generated by tandem mass spectrometry ofthe peptide ion 484.72 isolated from infected soluble B*0702 SupT1cells.

FIG. 9. Results of a PubMed BLAST search with the sequence GPRTAALGLLidentified in FIG. 8.

FIG. 10. Summary of Results of Entrez-PubMed search for the word“reticulocalbin”.

FIG. 11. Results of a peptide-binding algorithm performed using Parker'sPrediction using the entire source protein, reticulocalbin, whichgenerates a list of peptides which are bound by the B*0702 HLA allele.

FIG. 12. Results of a peptide-binding algorithm performed usingRammensee's SYPEITHI Prediction using the entire source protein,reticulocalbin, which generates a list of peptides which are bound bythe B*0702 HLA allele.

FIG. 13. Results of a predicted proteasomal cleavage of the completereticulocalbin protein using the cleavage predictor PaProC.

FIG. 14. Results of a predicted proteasomal cleavage of the completereticulocalbin protein using the cleavage predictor NetChop 2.0.

FIG. 15. Several high affinity peptides deriving from reticulocalbinwere identified as peptides predicted to be presented by HLA-A*0201 andA*0101.

FIG. 16. MS ion maps from soluble B*0702 uninfected SupT1 cells offractions 29 and 31 to determine that ion 484.72 was not present.

FIG. 17. Fragmentation patterns of soluble B*0702 uninfected SupT1 cellsfraction 30 ion 484.72 under identical MS collision conditions toillustrate the absence of the reticulocalbin peptide in the uninfectedcells.

FIG. 18. Comparison of the MS/MS fragmentation patterns of syntheticpeptide GPRTAALGLL and peptide ion 484.72 isolated from infected solubleB*0702 SupT1 cells.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail byway of exemplary drawings, experimentation, results, and laboratoryprocedures, it is to be understood that the invention is not limited inits application to the details of construction and the arrangement ofthe components set forth in the following description or illustrated inthe drawings, experimentation and/or results. The invention is capableof other embodiments or of being practiced or carried out in variousways. As such, the language used herein is intended to be given thebroadest possible scope and meaning; and the embodiments are meant to beexemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

The present invention generally relates to a method of epitope discoveryand comparative ligand mapping as well as methods of distinguishinginfected/tumor cells from uninfected/non-tumor cells. The present methodbroadly includes the following steps: (1) providing a cell linecontaining a construct that encodes an individual soluble class I orclass II MHC molecule (wherein the cell line is capable of naturallyprocessing self or nonself proteins into peptide ligands capable ofbeing loaded into the antigen binding grooves of the class I or class IIMHC molecules); (2) culturing the cell line under conditions which allowfor expression of the individual soluble class I or class II MHCmolecule from the construct, with such conditions also allowing for theendogenous loading of a peptide ligand (from the self or non-selfprocessed protein) into the antigen binding groove of each individualsoluble class I or class II MHC molecule prior to secretion of thesoluble class I or class II MHC molecules having the peptide ligandsbound thereto; and (4) separating the peptide ligands from theindividual soluble class I or class II MHC molecules.

The methods of the present invention may, in one embodiment, utilize amethod of producing MHC molecules (from genomic DNA or cDNA) that aresecreted from mammalian cells in a bioreactor unit. Substantialquantities of individual MHC molecules are obtained by modifying class Ior class II MHC molecules so that they are capable of being secreted,isolated, and purified. Secretion of soluble MHC molecules overcomes thedisadvantages and defects of the prior art in relation to the quantityand purity of MHC molecules produced. Problems of quantity are overcomebecause the cells producing the MHC do not need to be detergent lysed orkilled in order to obtain the MHC molecule. In this way the cellsproducing secreted MHC remain alive and therefore continue to produceMHC. Problems of purity are overcome because the only MHC moleculesecreted from the cell is the one that has specifically been constructedto be secreted. Thus, transfection of vectors encoding such secreted MHCmolecules into cells which may express endogenous, surface bound MHCprovides a method of obtaining a highly concentrated form of thetransfected MHC molecule as it is secreted from the cells. Greaterpurity is assured by transfecting the secreted MHC molecule into MHCdeficient cell lines.

Production of the MHC molecules in a hollow fiber bioreactor unit allowscells to be cultured at a density substantially greater thanconventional liquid phase tissue culture permits. Dense culturing ofcells secreting MHC molecules further amplifies the ability tocontinuously harvest the transfected MHC molecules. Dense bioreactorcultures of MHC secreting cell lines allow for high concentrations ofindividual MHC proteins to be obtained. Highly concentrated individualMHC proteins provide an advantage in that most downstream proteinpurification strategies perform better as the concentration of theprotein to be purified increases. Thus, the culturing of MHC secretingcells in bioreactors allows for a continuous production of individualMHC proteins in a concentrated form.

The method of producing MHC molecules utilized in the present inventionbegins by obtaining genomic or complementary DNA which encodes thedesired MHC class I or class II molecule. Alleles at the locus whichencode the desired MHC molecule are PCR amplified in a locus specificmanner. These locus specific PCR products may include the entire codingregion of the MHC molecule or a portion thereof. In one embodiment anested or hemi-nested PCR is applied to produce a truncated form of theclass I or class II gene so that it will be secreted rather thananchored to the cell surface. In another embodiment the PCR willdirectly truncate the MHC molecule.

Locus specific PCR products are cloned into a mammalian expressionvector and screened with a variety of methods to identify a cloneencoding the desired MHC molecule. The cloned MHC molecules are DNAsequenced to insure fidelity of the PCR. Faithful truncated clones ofthe desired MHC molecule are then transfected into a mammalian cellline. When such cell line is transfected with a vector encoding arecombinant class I molecule, such cell line may either lack endogenousclass I MHC molecule expression or express endogenous class I MHCmolecules. One of ordinary skill of the art would note the importance,given the present invention, that cells expressing endogenous class IMHC molecules may spontaneously release MHC into solution upon naturalcell death. In cases where this small amount of spontaneously releasedMHC is a concern, the transfected class I MHC molecule can be “tagged”such that it can be specifically purified away from spontaneouslyreleased endogenous class I molecules in cells that express class Imolecules. For example, a DNA fragment encoding a HIS tail may beattached to the protein by the PCR reaction or may be encoded by thevector into which the PCR fragment is cloned, and such HIS tail,therefore, further aids in the purification of the class I MHC moleculesaway from endogenous class I molecules. Tags beside a histidine tailhave also been demonstrated to work, and one of ordinary skill in theart of tagging proteins for downstream purification would appreciate andknow how to tag a MHC molecule in such a manner so as to increase theease by which the MHC molecule may be purified.

Cloned genomic DNA fragments contain both exons and introns as well asother non-translated regions at the 5′ and 3′ termini of the gene.Following transfection into a cell line which transcribes the genomicDNA (gDNA) into RNA, cloned genomic DNA results in a protein productthereby removing introns and splicing the RNA to form messenger RNA(mRNA), which is then translated into an MHC protein. Transfection ofMHC molecules encoded by gDNA therefore facilitates reisolation of thegDNA, mRNA/cDNA, and protein. Production of MHC molecules innon-mammalian cell lines such as insect and bacterial cells requirescDNA clones, as these lower cell types do not have the ability to spliceintrons out of RNA transcribed from a gDNA clone. In these instances themammalian gDNA transfectants of the present invention provide a valuablesource of RNA which can be reverse transcribed to form MHC cDNA. ThecDNA can then be cloned, transferred into cells, and then translatedinto protein. In addition to producing secreted MHC, such gDNAtransfectants therefore provide a ready source of mRNA, and thereforecDNA clones, which can then be transfected into non-mammalian cells forproduction of MHC. Thus, the present invention which starts with MHCgenomic DNA clones allows for the production of MHC in cells fromvarious species.

A key advantage of starting from gDNA is that viable cells containingthe MHC molecule of interest are not needed. Since all individuals inthe population have a different MHC repertoire, one would need to searchmore than 500,000 individuals to find someone with the same MHCcomplement as a desired individual—such a practical example of thisprinciple is observed when trying to find a donor to match a recipientfor bone marrow transplantation. Thus, if it is desired to produce aparticular MHC molecule for use in an experiment or diagnostic, a personor cell expressing the MHC allele of interest would first need to beidentified. Alternatively, in the method of the present invention, onlya saliva sample, a hair root, an old freezer sample, or less than amilliliter (0.2 ml) of blood would be required to isolate the gDNA.Then, starting from gDNA, the MHC molecule of interest could be obtainedvia a gDNA clone as described herein, and following transfection of suchclone into mammalian cells, the desired protein could be produceddirectly in mammalian cells or from cDNA in several species of cellsusing the methods of the present invention described herein.

Current experiments to obtain an MHC allele for protein expressiontypically start from mRNA, which requires a fresh sample of mammaliancells that express the MHC molecule of interest. Working from gDNA doesnot require gene expression or a fresh biological sample. It is alsoimportant to note that RNA is inherently unstable and is not as easilyobtained as is gDNA. Therefore, if production of a particular MHCmolecule starting from a cDNA clone is desired, a person or cell linethat is expressing the allele of interest must traditionally first beidentified in order to obtain RNA. Then a fresh sample of blood or cellsmust be obtained; experiments using the methodology of the presentinvention show that ≧5 milliliters of blood that is less than 3 days oldis required to obtain sufficient RNA for MHC cDNA synthesis. Thus, bystarting with gDNA, the breadth of MHC molecules that can be readilyproduced is expanded. This is a key factor in a system as polymorphic asthe MHC system; hundreds of MHC molecules exist, and not all MHCmolecules are readily available. This is especially true of MHCmolecules unique to isolated populations or of MHC molecules unique toethnic minorities. Starting class I or class II MHC molecule expressionfrom the point of genomic DNA simplifies the isolation of the gene ofinterest and insures a more equitable means of producing MHC moleculesfor study; otherwise, one would be left to determine whose MHC moleculesare chosen and not chosen for study, as well as to determine whichethnic population from which fresh samples cannot be obtained andtherefore should not have their MHC molecules included in a diagnosticassay.

While cDNA may be substituted for genomic DNA as the starting material,production of cDNA for each of the desired HLA class I types willrequire hundreds of different, HLA typed, viable cell lines, eachexpressing a different HLA class I type. Alternatively, fresh samplesare required from individuals with the various desired MHC types. Theuse of genomic DNA as the starting material allows for the production ofclones for many HLA molecules from a single genomic DNA sequence, as theamplification process can be manipulated to mimic recombinatorial andgene conversion events. Several mutagenesis strategies exist whereby agiven class I gDNA clone could be modified at either the level of gDNAor at the cDNA resulting from this gDNA clone. The process of producingMHC molecules utilized in the present invention does not require viablecells, and therefore the degradation which plagues RNA is not a problem.

The soluble class I MHC proteins produced by the method described hereinare utilized in the methods of epitope discovery and comparative ligandmapping of the present invention. The methods of epitope discovery andcomparative ligand mapping described herein which utilize secretedindividual MHC molecules have several advantages over the prior art,which utilized MHC from cells expressing multiple membrane-bound MHCs.While the prior art method could distinguish if an epitope was presentedon the surface of a cell, this prior art method is unable to directlydistinguish in which specific MHC molecule the peptide epitope wasbound. Lengthy purification processes might be used to try and obtain asingle MHC molecule, but doing so limits the quantity and usefulness ofthe protein obtained. The novelty and flexibility of the currentinvention is that individual MHC specificities can be utilized insufficient quantity through the use of recombinant, soluble MHCproteins.

Class I and class II MHC molecules are really a trimolecular complexconsisting of an alpha chain, a beta chain, and the alpha/beta chain'speptide cargo (i.e., peptide ligand) which is presented on the cellsurface to immune effector cells. Since it is the peptide cargo, and notthe MHC alpha and beta chains, which marks a cell as infected,tumorigenic, or diseased, there is a great need to identify andcharacterize the peptide ligands bound by particular MHC molecules. Forexample, characterization of such peptide ligands greatly aids indetermining how the peptides presented by a person with MHC-associateddiabetes differ from the peptides presented by the MHC moleculesassociated with resistance to diabetes. As stated above, having asufficient supply of an individual MHC molecule, and therefore that MHCmolecule's bound peptides, provides a means for studying such diseases.Because the method of the present invention provides quantities of MHCprotein previously unobtainable, unparalleled studies of MHC moleculesand their important peptide cargo can now be facilitated.

Therefore, the present invention is also related to methods of epitopediscovery and comparative ligand mapping which can be utilized todistinguish infected/tumor cells from uninfected/non-tumor cells byunique epitopes presented by MHC molecules in the disease or non-diseasestate.

Creation of sHLA Molecules from Genomic DNA (gDNA)

1. Genomic DNA Extraction. 200 ìl of sample either blood, plasma, serum,buffy coat, body fluid or up to 5×10⁶ lymphocytes in 200 ìl Phosphatebuffered saline were used to extract genomic DNA using the QIAamp® DNABlood Mini Kit blood and body fluid spin protocol. Genomic DNA qualityand quantity was assessed using optical density readings at 260 nm and280 nm.

TABLE I Primer name Sequence 5′-3′ Locus Cut site Annealing s

PP5UTA GCGCTCTAGACCCAGACGCCGAGGATGGCC A XbaI 5UT 3PPI4AGCCCTGACCCTGCTAAAGGT A Intron 4 PP5UTB GCGCTCTAGACCACCCGGACTCAGAATCTCCTB XbaI 5UT 3PPI4B TGCTTTCCCTGAGAAGAGAT B Intron 4 5UTB39AGGCGAATTCCAGAGTCTCCTCAGACGCG B*39 EcoRI 5UT B39 5PKCEGGGCGAATTCCCGCCGCCACCATGCGGGTCATGGCGCC C EcoRI 5UT 3PPI4CTTCTGCTTTCCTGAGAAGAC C Intron 4 PP5UTGGGCGAATTCGGACTCAGAATCTCCCCAGACGCCGAG B EcoRI 5UT PP3PEICCGCGAATTCTCATCTCAGGGTGAGGGGCT A, B, C EcoRI Exon 4 PP3PEIHCCGCAAGCTTTCATCTCAGGGTGAGGGGCT A, B, C HindIII Exon 4 3PEIHC7CCGCAAGCTTTCAGCTCAGGGTGAGGGGCT Cw*07 HindIII Exon 4

indicates data missing or illegible when filed

2.1 PCR Strategy. Primers were designed for HLA-A, -B and -C loci inorder to amplify a truncated version of the human class I MHC using a 2stage PCR strategy. The first stage PCR uses a primer set that amplifyfrom the 5′ Untranslated region to Intron 4. This amplicon is used as atemplate for the second PCR which results in a truncated version of theMHC Class I gene by utilizing a 3′ primer that sits down in exon 4, the5′ primer remains the same as the 1^(st) PCR. An overview can be seen inFIG. 1. The primers for each locus are listed in TABLE I. DifferentHLA-B locus alleles require primers with different restriction cut sitesdepending on the nucleotide sequence of the allele. Hence there are two5′ and two 3′ truncating primers for the −B locus.

2.2 Primary PCR. Materials: An Eppendorf Gradient Mastercycler is usedfor all PCR. (1) H₂O:Dionized ultrafiltered water (DIUF) FisherScientific, W2-4,41. (2) PCR nucleotide mix (10 mM eachdeoxyribonucleoside triphosphate [dNTP]), Boehringer Manheim, #1814,362. (3) 10×Pfx Amplification buffer, pH 9.0, GibcoBRL®, part #52806,formulation is proprietary information. (4) 50 mM MgSO₄, GibcoBRL®, part#52044 (5) Platinum® Pfx DNA Polymerase (B Locus only), GibcoBRL®,11708-013. (6) Pfu DNA Polymerase (A and C Locus), Promega, M7741. (7)Pfu DNA Polymerase 10× reaction Buffer with MgSO₄, 200 mM Tris-HCL, pH8.8, 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1 mg/ml nuclease freeBSA, 1% Triton® X-100. (8) Amplification primers (in ng/ìl) (see TABLEI): A locus: 5′ sense PP5UTA (300); 3′ antisense PPI4A (300); B locus(Not B*39's): sense PP5UTB (300); antisense PPI4B (300); B locus(B*39's): sense 5UTB39 (300); antisense PPI4B (300); C Locus: sense5PKCE (300); antisense PPI4C (300). (9) gDNA Template.

2.3 Secondary PCR (also used for colony PCR). (1) H₂O:Dionizedultrafiltered water (DIUF) Fisher Scientific, W2-4,41. (2) PCRnucleotide mix (10 mM each deoxyribonucleoside triphosphate [dNTP]),Boehringer Manheim, #1814, 362. (3) Pfu DNA Polymerase (A and C Locus),Promega, M7741. (4) Pfu DNA Polymerase 10× reaction Buffer with MgSO₄,200 mM Tris-HCL, pH 8.8, 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1mg/ml nuclease free BSA, 1% Triton® X-100. (5) Amplification primers (inng/ìl) see TABLE I: A-locus: 5′ sense PP5UTA (300), 3′ antisense PP3PEI(300); B-locus: sense PP5UTB (300), antisense PP3PEI (300); B-locus:sense PP5UT (300), antisense PP3PEIH (300); B-locus B39's: sense 5UTB39(300), antisense PP3PEIH (300); C-locus: sense 5PKCE (300), antisensePP3PEI (300); C-locus Cw*7's: sense 5PKCE (300), antisense 3PEIHC7(300). (6) Template 1:100 dilution of the primary PCR product.

2.4 Gel Purification of PCR products and vectors. (1) Dark ReaderTansilluminator Model DR-45M, Clare Chemical Research. (2) SYBR Green,Molecular Probes Inc. (3) Quantum Prep Freeze 'N Squeeze DNA GelRxtraction Spin Columns, Bio-Rad Laboratories, 732-6165.

2.5 Restriction digests, Ligation and Transformation. (1) Restrictionenzymes from New England Biolabs: (a) EcoR I #R0101S; (b) Hind III#R0104S; (c) Xba I #R0145S. (2) T4 DNA Ligase, New England Biolabs,#M0202S. (3) pcDNA3.1(−), Invitrogen Corporation, V795-20. (4) 10×Buffers from New England Biolabs: (a) EcoR I buffer, 500 mM NaCl, 1000mM Tris-HCL, 10 mM MgCL₂, 0.25% Triton-X 100, pH 7.5; (b) T4 DNA ligasebuffer, 500 mM Tris-HCL, 100 mM MgCL₂, 100 mM DTT, 10 mM ATP, 250 ug/mlBSA, pH 7.5; (c) NEB buffer 2, 500 mM NaCl, 100 mM Tris-HCl, 100 mMMgCl₂, 10 mM DDT, pH 7.9. (5) 100×BSA, New England Biolabs. (6)Z-Competent E. coli Transformation Buffer Set, Zymo Research, T3002. (7)E. coli strain JM109. (8) LB Plates with 100 ìg/ml ampicillin. (9) LBmedia with 100 ìg/ml ampicillin.

2.6 Plasmid Extraction. Wizard Plus SV minipreps, Promega, #A1460.

2.7 Sequencing of Clones. (1) Thermo Sequenase Primer Cycle SequencingKit, Amersham Pharmacia Biotech, 25-2538-01. (2) CY5 labelled primers(see TABLE II). (3) AlfExpress automated DNA sequencer, AmershamPharmacia Biotech.

TABLE II Primer Name Sequence 5′-3′ Seq. ID NO: T7PromTAATACGACTCACTATAGGG 12 BGHrev TAGAAGGCACAGTCGAGG 13 PPI2E2RGTCGTGACCTGCGCCCC 14 PPI2E2F TTTCATTTTCAGTTTAGGCCA 15 ABCI3E4FGGTGTCCTGTCCATTCTCA 16

2.8 Gel Casting. (1) PagePlus 40% concentrate, Amresco, E562, 500 ml.(2) Urea, Amersham Pharmacia Biotech, 17-0889-01,500g. (3) 3N′N′N′N′-tetramethylethyleneiamine (TEMED), APB. (4) Ammoniumpersulphate (10% solution), APB. (5) Boric acid, APB. (6) EDTA-disodiumsalt, APB. (7) Tris, APB. (8) Bind-Saline, APB. (9) Isopropanol, Sigma.(10) Glacial Acetic Acid, Fisher Biotech. (11) DIUF water, FisherScientific. (12) EtOH 200-proof.

2.9 Plasmid Preparation for Electroporation. Qiagen Plasmid Midi kit,Qiagen Inc., 12143.

3.0 Electroporation. (1) Biorad Gene Pulser with capacitance extender,Bio-Rad Laboratories. (2) Gene Pulser Cuvette, Bio-Rad Laboratories. (3)Cytomix: 120 mM KCl, 0.15 mM CaCl₂, 10 mMK₂HPO₄/KH₂PO₄, pH 7.6, 25 mMHepes, pH 7.6, 2 mM EGTA, pH 7.6, 5 mM MgCl₂, pH 7.6 with KOH. (4) RPMI1640+ 20% Foetal Calf Serum+Pen/strep. (5) Haemacytometer. (6) LightMicroscope. (7) CO₂ 37° Incubator. (8) Cells in log phase.

Primary PCR

A-Locus and C-Locus

10x Pfu buffer 5 μl 5′ Primer (300 ng/μl) 1 μl 3′ Primer (300 ng/μl) 1μl dNTP's (10 mM each) 1 μl gDNA (50 ng/μl) 10 μl  DIUF H₂0 31.4 μl  Pfu DNA Polymerase 0.6 μl   96° C.  2 min. 95° C. 1 min 58° C. 1 min{close oversize brace} x35 73° C. 5 min 73° C. 10 min 

B-Locus

10x Pfx buffer 5 μl 5′ Primer (300 ng/μl) 1 μl 3′ Primer (300 ng/μl) 1μl dNTP's (10 mM each) 1.5 μl   MgSO₄ (50 mM) 1 μl gDNA (100 ng/μl) 1 μlDIUF H₂O 40 μl  Pfx DNA Polymerase 0.5 μl   94° C.  2 min. 94° C. 1 min60° C. 1 min {close oversize brace} x35 68° C. 3.5 min   68° C. 5 min

Gel Purification of PCR (all PCR and Plasmids are Gel Purified)

Mix primary PCR with 5 ìl of 1×SYBR green and incubate at roomtemperature for 15 minutes then load on a 1% agarose gel. Visualize onthe Dark Reader and purify using the Quantum Prep Freeze and Squeezeextraction kit according to the manufacturers instructions.

Secondary PCR

A, B and C Loci

10x Pfu buffer   5 μl 5′ Primer (300 ng/μl) 0.5 μl 3′ Primer (300 ng/μl)0.5 μl dNTP's (10 mM each)   1 μl 1:100 1° PCR  10 μl DIUF H₂0 37.5 μl Pfu DNA Polymerase 0.5 μl 96° C.  2 min. 95° C. 1 min 58° C. 1 min{close oversize brace} x35 73° C. 4 min 73° C. 7 min

Restriction Digests

2° PCR (gel purified) 30 μl Restriction enzyme 1 X μl Restriction enzyme2 X μl 10x buffer 5 μl 100x BSA 0.5 μl DIUF H₂O 10.5 μl

The enzymes used will be determined by the cut sites incorporated intothe PCR primers for each individual PCR. The expression vector pcDNA3.1(−) will be cut in a similar manner.

Ligation

PcDNA3.1(—) cut with same enzymes as PCR 50 ng Cut PCR 100 ng 10x T4 DNAligase buffer 2 μl T4 DNA Ligase 1 μl DIUF H₂0 up to 20 μl

Transformation

Transform JM109 using competent cells made using Z-competent E. coliTransformation Kit and Buffer Set and follow the manufacturersinstructions.

Colony PCR

This will check for insert in any transformed cells. Follow the sameprotocol for the secondary PCR.

Mini Preps of Colonies with Insert

Use the Wizard Plus SV minipreps and follow the manufacturersinstructions. Make glycerol stocks before beginning extraction protocol.

Sequencing of Positive Clones

Using the Thermo Sequenase Primer Cycle Sequencing Kit

A, C, G or T mix 3 μl CY5 Primer 1 pm/μl 1 μl DNA template 100 ng/μl 5μl 96° C.  2 min 96° C. 30 sec {close oversize brace} x25 61° C. 30 sec

Add 6 ìl formamide loading buffer and load 10 ìl onto sequencing gel.Analyse sequence for good clones with no misincorporations.

Midi Preps

Prepare plasmid for electroporation using the Qiagen Plasmid Midi Kitaccording to the manufacturers instructions.

Electroporation

Electroporations are performed as described in “The Bw4 public epitopeof HLA-B molecules confers reactivity with natural killer cell clonesthat express NKb1, a putative HLA receptor. Gumperz, J. E., V. Litwin,J. H. Phillips, L. L. Lanier and P. Parham. J. Exp. Med. 181:1133-1144,1995, which is expressly incorporated herein by reference.”

Screening for Production of Soluble HLA

An ELISA is used to screen for the production of soluble HLA. Forbiochemical analysis, monomorphic monoclonal antibodies are particularlyuseful for identification of HLA locus products and their subtypes.

W6/32 is one of the most common monoclonal antibodies (mAb) used tocharacterize human class I major histocompatibility complex (MHC)molecules. It is directed against monomorphic determinants on HLA-A, -Band -C HCs, which recognizes only mature complexed class I molecules andrecognizes a conformational epitope on the intact MHC moleculecontaining both beta2-microglobulin (atm) and the heavy chain (HC).W6/32 binds a compact epitope on the class I molecule that includes bothresidue 3 of beta2m and residue 121 of the heavy chain (Ladasky J J,Shum B P, Canavez F, Seuanez H N, Parham P. Residue 3 ofbeta2-microglobulin affects binding of class I MHC molecules by theW6/32 antibody. Immunogenetics 1999 April; 49(4):312-20.). The constantportion of the molecule W6/32 binds to is recognized by CTLs and thuscan inhibit cytotoxicity. The reactivity of W6/32 is sensitive to theamino terminus of human beta2-microglobulin (Shields M J, Ribaudo R K.Mapping of the monoclonal antibody W6/32: sensitivity to the aminoterminus of beta2-microglobulin. Tissue Antigens 1998 May,51(5):567-70). HLA-C could not be clearly identified inimmunoprecipitations with W6/32 suggesting that HLA-C locus products maybe associated only weakly with b2m, explaining some of the difficultiesencountered in biochemical studies of HLA-C antigens [Stam, 1986 #1].The polypeptides correlating with the C-locus products are recognizedfar better by HC-10 than by W6/32 which confirms that at least some ofthe C products may be associated with b2m more weakly than HLA-A and -B.W6/32 is available biotinylated (Serotec MCA81B) offering additionalvariations in ELISA procedures.

HC-10 is reactive with almost all HLA-B locus free heavy chains. The A2heavy chains are only very weakly recognized by HC-10. Moreover, HC-10reacts only with a few HLA-A locus heavy chains. In addition, HC-10seems to react well with free heavy chains of HLA-C types. No evidencefor reactivity of HC-10 with heavy-chain/b2m complex has been obtained.None of the immunoprecipitates obtained with HC-10 contained b2m [Stam,1986 #1]. This indicates that HC-10 is directed against a site of theHLA class I heavy chain that includes the portion involved ininteraction with the atm. The pattern of HC-10 precipitated material isqualitatively different from that isolated with W6/32.

TP25.99 detects a determinant in the alpha3 domain of HLA-ABC. It isfound on denatured HLA-B (in Western) as well as partially or fullyfolded HLA-A, B, & C. It doesn't require a peptide or â2m, i.e., itworks with the alpha 3 domain which folds without peptide. This makes ituseful for HC determination.

Anti-human â2m (HRP) (DAKO P0174) recognizes denatured as well ascomplexed â2m. Although in principle anti-â2m reagents could be used forthe purpose of identification of HLA molecules, they are less suitablewhen association of heavy chain and â2m is weak. The patterns of class Imolecules precipitated with W6/32 and anti-â2m are usuallyindistinguishable [Vasilov, 1983 #10].

Rabbit anti-â2-microglobulin dissociates â2-microglobulin from heavychain as a consequence of binding (Rogers, M. J., Appella, E., Pierotti,M. A., Invernizzi, G., and Parmiani, G. (1979) Proc Natl. Acad. Sci.U.S.A. 76, 1415-1419). It also has been reported that rabbit anti-humanâ2-microglobulin dissociactes â2-microglobulin from HLA heavy chainsupon binding (Nakamuro, K., Tanigaki, N., and Pressman, D. (1977)Immunology 32, 139-146.). This anti-human â2m antibody is also availableunconjugated (DAKO A0072).

The W6/32-HLA sandwich ELISA. Sandwich assays can be used to study anumber of aspects of protein complexes. If antibodies are available todifferent components of a heteropolymer, a two-antibody assay can bedesigned to test for the presence of the complex. Using a variation ofthese assays, monoclonal antibodies can be used to test whether a givenantigen is multimeric. If the same monoclonal antibody is used for boththe solid phase and the label, monomeric antigens cannot be detected.Such combinations, however, may detect multimeric forms of the antigen.In these assays negative results may be generated both by multimericantigen held in unfavorable steric positions as well as by monomericantigens.

The W6/32—anti-â2m antibody sandwich assay is one of the best techniquesfor determining the presence and quantity of sHLA. Two antibody sandwichassays are quick and accurate, and if a source of pure antigen isavailable, the assay can be used to determine the absolute amounts ofantigen in unknown samples. The assay requires two antibodies that bindto non-overlapping epitopes on the antigen. This assay is particularlyuseful to study a number of aspects of protein complexes.

To detect the antigen (sHLA), the wells of microtiter plates are coatedwith the specific (capture) antibody W6/32 followed by the incubationwith test solutions containing antigen. Unbound antigen is washed outand a different antigen-specific antibody (anti-â2m) conjugated to HRPis added, followed by another incubation. Unbound conjugate is washedout and substrate is added. After another incubation, the degree ofsubstrate hydrolysis is measured. The amount of substrate hydrolyzed isproportional to the amount of antigen in the test solution.

The major advantages of this technique are that the antigen does notneed to be purified prior to use and that the assays are very specific.The sensitivity of the assay depends on 4 factors: (1) The number ofcapture antibody; (2) The avidity of the capture antibody for theantigen; (3) The avidity of the second antibody for the antigen; (4) Thespecific activity of the labeled second antibody.

Using an ELISA protocol template and label a clear 96-well polystyreneassay plate. Polystyrene is normally used as a microtiter plate.(Because it is not translucent, enzyme assays that will be quantitatedby a plate reader should be performed in polystyrene and not PVCplates).

Coating of the W6/32 is performed in Tris buffered saline (TBS); pH 8.5.A coating solution of 8.0 ìg/ml of specific W6/32 antibody in TBS (pH8.5) is prepared. (blue tube preparation stored at −20 C with aconcentration of 0.2 mg/ml and a volume of 1 ml giving 0.2 mg per tube).

TABLE III No. of Total W6/32 TBS plates Volume antibody pH 8.5 1 10 ml 400 μl  9.6 ml 2 20 ml  800 μl 19.2 ml 3 30 ml 1200 μl 28.8 ml 4 40 ml1600 μl 38.4 ml 5 50 ml 2000 μl 48.0 ml

Although this is well above the capacity of a microtiter plate, thebinding will occur more rapidly. Higher concentrations will speed thebinding of antigen to the polystyrene but the capacity of the plastic isonly about 100 ng/well (300 ng/cm₂), so the extra protein will not bind.(If using W6/32 of unknown composition or concentration, first titratethe amount of standard antibody solution needed to coat the plate versusa fixed, high concentration of labeled antigen. Plot the values andselect the lowest level that will yield a strong signal. Do not includesodium azide in any solutions when horseradish peroxidase is used fordetection.

Immediately coat the microtiter plate with 100 ìl of antigen solutionper well using a multichannel pipet. Standard polystyrene will bindantibodies or antigens when the proteins are simply incubated with theplastic. The bonds that hold the proteins are non-covalent, but theexact types of interactions are not known. Shake the plate to ensurethat the antigen solution is evenly distributed over the bottom of eachwell. Seal the plate with plate sealers (sealplate adhesive sealingfilm, nonsterile, 100 per unit; Phenix; LMT-Seal-EX) or sealing tape toNunc-Immuno™ Modules (#236366). Incubate at 4° C. overnight. Avoiddetergents and extraneous proteins. Next day, remove the contents of thewell by flicking the liquid into the sink or a suitable waste container.Remove last traces of solution by inverting the plate and blotting itagainst clean paper toweling. Complete removal of liquid at each step isessential for good performance.

Wash the plate 10 times with Wash Buffer (PBS containing 0.05% Tween-20)using a multi-channel ELISA washer. After the last wash, remove anyremaining Wash Buffer by inverting the plate and blotting it againstclean paper toweling. After the W6/32 is bound, the remaining sites onthe plate must be saturated by incubating with blocking buffer made of3% BSA in PBS. Fill the wells with 20011 blocking buffer. Cover theplates with an adhesive strip and incubate overnight at 4° C.Alternatively, incubate for at least 2 hours at room temperature whichis, however, not the standard procedure. Blocked plates may be storedfor at least 5 days at 4° C. Good pipetting practice is most importantto produce reliable quantitative results. The tips are just as importanta part of the system as the pipette itself. If they are of inferiorquality or do not fit exactly, even the best pipette cannot producesatisfactory results. The pipette working position is always vertical:Otherwise causing too much liquid to be drawn in. The immersion depthshould be only a few millimeters. Allow the pipetting button to retractgradually, observing the filling operation. There should be noturbulence developed in the tip, otherwise there is a risk of aerosolsbeing formed and gases coming out of solution.

When maximum levels of accuracy are stipulated, prewetting should beused at all times. To do this, the required set volume is first drawn inone or two times using the same tip and then returned. Prewetting isabsolutely necessary on the more difficult liquids such as 3% BSA. Donot prewet, if your intention is to mix your pipetted sample thoroughlywith an already present solution. However, prewet only for volumesgreater than 10 ìl. In the case of pipettes for volumes less than 10 ìlthe residual liquid film is as a rule taken into account when designingand adjusting the instrument. The tips must be changed between eachindividual sample. With volumes<10 ìl special attention must also bepaid to drawing in the liquid slowly, otherwise the sample will besignificantly warmed up by the frictional heat generated. Then slowlywithdraw the tip from the liquid, if necessary wiping off any dropsclinging to the outside.

To dispense the set volume hold the tip at a slight angle, press it downuniformly as far as the first stop. In order to reduce the effects ofsurface tension, the tip should be in contact with the side of thecontainer when the liquid is dispensed. After liquid has been dischargedwith the metering stroke, a short pause is made to enable the liquidrunning down the inside of the tip to collect at its lower end. Thenpress it down swiftly to the second stop, in order to blow out the tipwith the extended stroke with which the residual liquid can be blownout. In cases that are not problematic (e.g., aqueous solutions) thisbrings about a rapid and virtually complete discharge of the set volume.In more difficult cases, a slower discharge and a longer pause beforeactuating the extended stroke can help. To determine the absolute amountof antigen (sHLA), sample values are compared with those obtained usingknown amounts of pure unlabeled antigen in a standard curve.

For accurate quantitation, all samples have to be run in triplicate, andthe standard antigen-dilution series should be included on each plate.Pipetting should be preformed without delay to minimize differences intime of incubation between samples. All dilutions should be done inblocking buffer. Thus, prepare a standard antigen-dilution series bysuccessive dilutions of the homologous antigen stock in 3% BSA in PBSblocking buffer. In order to measure the amount of antigen in a testsample, the standard antigen-dilution series needs to span most of thedynamic range of binding. This range spans from 5 to 100 ng sHLA/ml. Astock solution Ê of 1 ìg/ml should be prepared, aliquoted in volumes of300 ìl and stored at 4 C. Prepare a 50 ml batch of standard at the time.(New batches need to be compared to the old batch before used inquantitation).

Use a tube of the standard stock solution E to prepare successivedilutions. While standard curves are necessary to accurately measure theamount of antigen in test samples, they are unnecessary for qualitative“yes/no” answers. For accurate quantitation, the test solutionscontaining sHLA should be assayed over a number of at least 4 dilutionsto assure to be within the range of the standard curve. Prepare serialdilutions of each antigen test solution in blocking buffer (3% BSA inPBS). After mixing, prepare all dilutions in disposable U-bottom 96 wellmicrotiter plates before adding them to the W6/32-coated plates with amultipipette. Add 150 ìl in each well. To further proceed, remove anyremaining blocking buffer and wash the plate as described above. Theplates are now ready for sample addition. Add 100 ìl of the sHLAcontaining test solutions and the standard antigen dilutions to theantibody-coated wells.

Cover the plates with an adhesive strip and incubate for exactly 1 hourat room temperature. After incubation, remove the unbound antigen bywashing the plate 10× with Wash Buffer (PBS containing 0.05% Tween-20)as described. Prepare the appropriate developing reagent to detect sHLA.Use the second specific antibody, anti-human atm-HRP (DAKO P0174/0.4mg/ml) conjugated to Horseradish Peroxidase (HRP). Dilute the anti-humanatm-HRP in a ratio of 1:1000 in 3% BSA in PBS. (Do not include sodiumazide in solutions when horseradish peroxidase is used for detection).

TABLE IV No. of Total anti-β2m-HRP 3% BSA plates Volume antibody in PBS1 10 ml 10 μl 10 ml 2 20 ml 20 μl  2 ml 3 30 ml 30 μl 30 ml 4 40 ml 40μl 40 ml 5 50 ml 50 μl 50 ml

Add 100 ìl of the secondary antibody dilution to each well. Alldilutions should be done in blocking buffer. Cover with a new adhesivestrip and incubate for 20 minutes at room temperature. Prepare theappropriate amount of substrate prior to the wash step. Bring thesubstrate to room temperature.

OPD (o-Phenylenediamine) is a peroxidase substrate suitable for use inELISA procedures. The substrate produces a soluble end product that isyellow in color. The OPD reaction is stopped with 3NH₂SO₄, producing anorange-brown product and read at 492 nm. Prepare OPD fresh from tablets(Sigma, P6787; 2 mg/tablet). The solid tablets are convenient to usewhen small quantities of the substrate are required. After secondantibody incubation, remove the unbound secondary reagent by washing theplate 10× with Wash Buffer (PBS containing 0.05% Tween-20). After thefinal wash, add 100 ìl of the OPD substrate solution to each well andallow to develop at room temperature for 10 minutes. Reagents of thedeveloping system are light-sensitive, thus, avoid placing the plate indirect light. Prepare the 3NH₂SO₄ stop solution. After 10 minutes, add100 ìl of stop solution per 100 ìl of reaction mixture to each well.Gently tap the plate to ensure thorough mixing.

Read the ELISA plate at a wavelength of 490 nm within a time period of15 minutes after stopping the reaction. The background should be around0.1. If your background is higher, you may have contaminated thesubstrate with a peroxidase. If the substrate background is low and thebackground in your assay is high, this may be due to insufficientblocking. Finally analyze your readings. Prepare a standard curveconstructed from the data produced by serial dilutions of the standardantigen. To determine the absolute amount of antigen, compare thesevalues with those obtained from the standard curve.

Creation of Transfectants and Production of Soluble Class I Molecules

Transfectants were established as previously described (Prilliman, K Ret al., Immunogenetics 45:379, 1997, which is expressly incorporatedherein by reference) with the following modifications: a cDNA clone ofB*1501 containing the entire coding region of the molecule was PCRamplified in order to generate a construct devoid of the cytoplasmicdomain using primers 5PXI (59-GGGCTCTAGAGGACTCAGAATCTCCCCAGAC GCCGAG-39)and 3PEI (59-CCGCGAATTCTCATCTCAGGGTGAG-39) as shown in TABLE V.Constructs were also created containing a C-terminal epitope tagconsisting of either 6 consecutive histidines or the FLAG epitope(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). TABLE V Primers utilized to createB*1501-HIS and B*1501-FLAG were 5PXI and3PEIHIS(59-CCGCGAATTCTCAGTGGTGGTGGTGGTGGTGCCATCTCAGGGTGAG-39) or3PEIFLAG (59-CCGCGAATTCTCACTTGTCATCGTCGTCCTTGTAATCCCATCTCAGGGTGAG-39).PCR amplicons were purified using a Qiagen Spin PCR purification kit(Qiagen, Levsden, The Netherlands) and cloned into the mammalianexpression vector pcDNA 3.1 (Invitrogen, Carlsbad, Calif., USA). TABLEV. After confirmation of insert integrity by bidirectional DNAsequencing, constructs were electroporated into the class I negativeB-lymphoblastoid cell line 721.221 (Prilliman, K R et al., 1997,previously incorporated herein by reference). Transfectants weremaintained in medium containing G418 post-electroporation and subclonedin order to isolate efficient producers of soluble class I as determinedby ELISA (Prilliman, K R et al, 1997, previously incorporated herein byreference).

TABLE V Full-length Seq. Primer or ID name Sequence Truncating Notes NO:HLA5UT GGGCGTCGACGGACTCAGAATCT either 5′ primer, SaI I cut site 17CCCCAGACGCCGAG 5UTA GCGCGTCGACCCCAGACGCCGAG either 5′primer, SaI I cut site A-locus specific 18 GATGGCC 5PXIGGGCTCTAGAGGACTCAGAATCT either 5′ primer, Xba I cut site 19CCCCAGACGCCGAG CLSP23 CCGCGTCGACTCAGATTCTCCCC full-length 5′primer, SaI I cut site C-locus specific 20 AGACGCCGAGATG LDC3UTACCGCAAGCTTAGAAACAAAGTCA full-length 3′ primer, HindIII cut siteA-locus specific 21 GGGTT CLSP1085 CCGCAAGCTTGGCAGCTGTCTCA full-length3′ primer, HindIII cut site C-locus specific 22 GGCTTTACAAG(CT)G 3UTACCGCAAGCTTTTGGGGAGGGAGC full-length 3′ primer, HindIII cut siteA-locus specific 23 ACAGGTCAGCGTGGGAAG 3UTB CCGCAAGCTTCTGGGGAGGAAACfull-length 3′ primer, HindIII cut site B-locus specific 24ATAGGTCAGCATGGGAAC 3PEI CCGCGAATTCTCATCTCAGGGTG truncating 3′primer, EcoRI cut site 25 AG 3PEIHIS CCGCGAATTCTCAGTGGTGGTGG truncating3′ primer, EcoRI cut site adds hexa- 26 TGGTGGTGCCATCTCAGGGTGAGhistidine tail 3PEIFLAG CCGCGATTCTCACTTGTCATCGT truncating 3′primer, EcoRI cut site adds FLAG- 27 CGTCCTTGTAATCCCATCTCAGG epitopeGTGAG 5PKOZXB GGGCTCTAGACCGCCGCCACCAT either 5′ primer, Xba I cut siteC-locus specific 28 GCGGGTCATGGCGCC

Soluble B*1501, B*1501-HIS, and B*1501-FLAG were produced by culturingestablished transfectants in CP3000 hollow-fiber bioreactors aspreviously described by Prilliman et al, 1997, which has previously beenincorporated herein by reference. Supernatants containing soluble classI molecules were collected in bioreactor harvests and purified on W6/32affinity columns. At least 2 column purifications were performed permolecule.

Ligand Purification, Edman Sequencing, and Reverse-Phase HPLC Separationof Peptides

Peptide ligands were purified from class I molecules by acid elution(Prilliman, K R et al., Immunogenetics 48:89, 1998 which is expresslyincorporated herein by reference) and further separated from heavy andlight chains by passage through a stirred cell (Millipore, Bedford,Mass., USA) equipped with a 3-Kd cutoff membrane (Millipore).Approximately 1/100 volume of stirred cell flow through containingpeptide eluted from either B*1501, B*1501-HIS, or B*1501-FLAG wassubjected to 14 cycles of Edman degradation on a 492A pulsed liquidphase protein sequencer (Perkin-Elmer Applied Biosystems Division,Norwalk, Conn., USA) without the derivitization of cysteine. Edmanmotifs were derived by combining from multiple column elutions thepicomolar yields of each amino acid and then calculating the foldincrease over previous round as described in (Prilliman, K R et al,1998, previously incorporated herein by reference) and are shown in FIG.2.

Pooled peptide eluate was separated into fractions by RP-HPLC aspreviously described (Prilliman, K R et al, 1998, previouslyincorporated herein by reference). Briefly, 400-mg aliquots of peptideswere dissolved in 100 ml of 10% acetic acid and loaded onto a 2.1 3 150mm C18 column (Michrom Bioresources, Auburn, Calif., USA) using agradient of 2%-10% acetonitrile with 0.06% TFA for 0.02 min followed bya 10%-60% gradient of the same for 60 min. Fractions were collectedautomatically at 1-min intervals with a flow rate of 180 ml/min.

Mass Spectrometric Ligand Analysis

RP-HPLC fractions were speed-vacuumed to dryness and reconstituted in 40ml 50% methanol, 0.5% acetic acid. Approximately 6 ml from selectedfractions were sprayed into an API-III triple quadrupole massspectrometer (PE Sciex, Foster City, Calif., USA) using a NanoESionization source inlet (Protana, Odense, Denmark). Scans were collectedwhile using the following instrument settings: polarity—positive; needlevoltage—1375 V; orifice voltage—65 V; N2 curtain gas—0.6 ml/min; stepsize—0.2 amu; dwell time—1.5 ms; and mass range—325-1400. Total iontraces generated from each molecule were compared visually in order toidentify ions overlapping between molecules. Following identification ofion matches, individual ions were selected for MS/MS sequencing.

Sequences were predicted using the BioMultiView program (PE Sciex)algorithm predict sequence, and fragmentation patterns further assessedmanually. Determinations of ion sequence homology to currently compiledsequences were performed using advanced BLAST searches against thenonredundant, human expressed sequence tag, and unfinished highthroughput genomic sequences nucleotide databases currently availablethrough the National Center for Biotechnology Information (NationalInstitutes of Health, Bethesda, Md., USA).

The methodology of the present invention provides a direct comparativeanalysis of peptide ligands eluted from class I HLA molecules. In orderto accomplish such comparative analyses, hollow-fiber bioreactors forclass I ligand production were used along with reverse-phase HPLC forfractionating eluted ligands, and mass spectrometry for the mapping andsequencing of peptide ligands. The application of comparative ligandmapping also is applicable to cell lines that express endogenous classI. Prior to peptide sequence determination in class I positive celllines, the effects of adding a C-terminal epitope tag to transfectedclass I molecules was found to have no deleterious effects. Either a tagconsisting of 6 histidines (6-HIS) or a tag containing the epitopeAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) was added to the C-terminus ofsoluble B*1501 through PCR. These constructs were then transfected intoclass I negative 721.221 cells and peptides purified as previouslyestablished (Prilliman, K R et al, 1998, previously incorporated hereinby reference). Comparison of the two tailed transfectants with theuntailed, soluble B*1501 allowed for the determination that tag additionhad no effect on peptide binding specificity of the class I molecule andconsequently had no deleterious effects on direct peptide ligand mappingand sequencing.

Edman Motifs

The most common means for discerning ligands presented by a particularclass I molecule is Edman sequencing the pool of peptides eluted fromthat molecule. In order to demonstrate that tailing class I moleculeswith Cterminal tags does not disrupt endogenous peptide loading, Edmansequences of the peptide pools from B*1501, B*1501-HIS, and B*1501-FLAGwas compared with previously published B*1501 data FIG. 2. Motifs wereassigned to each of the various B*1501 molecules as shown in FIG. 2. Atthe anchor position 2 (P2) a dominant Q and subdominant M was seen inmotifs as previously published by Falk et al. (Immunogenetics 41:165,1995) and Barber et al. (J Exp Med 184:735, 1996). A more disparate P3is seen in all molecules with F, K, N, P, R, and Y appearing; theseresults have also been previously reported by Falk and Barber. Again, adominant Y and F are seen as the C-terminal anchors at P9 in all threemolecules. The motif data for all three molecules are in close accord,therefore, with the published standard motifs.

Mass Spectrometric Profiles

Comparison of motifs for the surface bound, nontailed, and tailed B*1501molecules identified no substantial differences in the pooled peptidesbound by the various forms of B*1501 tested. However, the aim of thepresent invention is to subtractively compare the individual peptidesbound by class I molecules from diseased and healthy cells. Subtractiveanalysis is accomplished through the comparison of mass spectrometricion maps and, as such, the ion maps of tailed and untailed class Imolecules were compared in order to determine the effect of tailing uponcomparative peptide mapping.

Peptides derived from tailed and untailed B*1501 were separated intofractions via reverse phase HPLC (RP-HPLC). Each fraction was thenscanned using an API-III mass spectrometer in order to identify ionspresent in each fraction. Overall ion scans from RP-HPLC fractions 9,10, 11, 18, 19, and 20 were produced and visually compared in order toassess ions representing peptides overlapping between the threemolecules. FIG. 3. depicts a representative section of the ion mapsgenerated from each of the molecules. This comparison shows that thesame pattern of ions is produced by the different B*1501 moleculesanalyzed here. The manual comparison of ion maps from each of the threefractions found little to no difference in the peptides bound by each ofthe three molecules.

Ligand Sequences

After identification of ion matches in MS chromatograms of each of thethree molecules, individual ions were chosen for sequencing by tandemmass spectrometry in order to determine if ions were indeed matched atthe peptide-sequence level. Ten ions from each fraction were initiallyselected for MS/MS sequence generation. Fragmentation patterns for eachof the ions from each molecule were manually compared and identicalfragmentation patterns were counted as peptide-sequence level matches,as illustrated in FIG. 4. Of the peptide fragmentation patternsexamined, 52/57 (91%) were exact matches between the untailed moleculesand the 6-HIStailed protein (TABLE VI). A more disparate pattern offragmentation was identified in the FLAG-tailed ions selected for MS/MSsequencing: of the 57 ions selected for MS/MS fragmentation comparison,39 (70%) fragmentation patterns matched between the FLAG-tailed anduntailed molecules. Overall, 91 out of 113 (81%) spectra examined werein accord between the tailed molecules and soluble B*1501.

TABLE VI Molecules Ions Examined Ion Matches Percent Matched B*1501-HIS57 52 91% B*1501-FLAG 56 39 70% B*1501-Tagged 113 91 81%

Several ligand sequences were clearly determined from the fragmentationpatterns produced. The ligand QGLISRGYSY, deriving from humanperiplakin, was sequenced from those peptides eluted in fraction 18. Asecond ligand, AVRDISEASVF, an 11-mer matching a span of the 40Sribosomal protein S26, was identified in fraction 20. Notably, these twopeptides lacked the strong consensus glutamine expected by the motifdata, a phenomenon previously reported by our laboratory when sequencingB*1501-eluted ligands (Prilliman, K R et al, 1997, previouslyincorporated herein by reference). Both these ligands, however,terminate with an aromatic tyrosine or phenylalanine; these amino acidswere both predicted to be strong anchors by Edman sequencing data and bypreviously published observations (Prilliman, K R et al, 1998,previously incorporated herein by reference).

One embodiment of the present invention contemplates characterizingpeptide ligands bound by a given class I molecule by transfecting thatmolecule into a class I negative cell line and affinity purification ofthe class I molecule and bound peptide. Complications arise, however,when cell lines are chosen for study that already possess class Imolecules. In this case, antibodies specific for one class I moleculemust be used to selectively purify that class I molecule from othersexpressed by the cell. Because allele-specific antibodies recognizeepitopes in and around the peptide binding groove, variations in thepeptides found in the groove can alter antibody affinity for the class Imolecule (Solheim, J C et al., J Immunol 151:5387, 1993; and Bluestone,J A et al., J Exp Med 176:1757, 1992). Altered antibody recognition can,in turn, bias the peptides available for elution and subsequent sequenceanalysis.

In order to selectively purify from a class I positive cell atransfected class I molecule and its peptide ligands in an unbiased way,it was necessary to alter the embodiment for class I purification in anon-class I positive cell. The C-terminal addition of a FLAG and 6-HIStag to a class I molecule that had already been extensivelycharacterized, B*1501 was shown to have little or no effect on peptidebinding. This methodology was designed to allow purification of a singleclass I specificity from a complex mixture of endogenously expressedclass I molecules. Ligands eluted from the tailed and untailed B*1501molecule were compared to assess the effect of a tail addition on thepeptide repertoire.

Pooled Edman sequencing is the commonly used method to determine thebinding fingerprint of a given molecule, and this methodology was usedto ascertain the large-scale effect of tail addition upon peptidebinding. We subjected 1/100 of the peptides eluted from each class I MHCmolecule to Edman degradation and derived motifs for each of themolecules. Both the HIS- and FLAG-tailed motifs matched published motifsfor the soluble and membrane-bound B*1501. Each of the moleculesexhibited motifs bearing a dominant P2 anchor of Q, a more disparate P3in which multiple residues could be found, and another dominant anchorof Y or F at P9. Small differences in the picomolar amounts of each ofthe amino acids detected during Edman sequencing have been notedpreviously in consecutive runs with the same molecule and most likelyreflect differences in cell handling and/or peptide isolation ratherthan disparities in bound peptides. Highly similar peptide motifsindicated that the peptide binding capabilities of class I MHC moleculesare not drastically altered by the addition of a tag.

In order to insure the ligands were not skewed after tag addition, MSand MS/MS were used for the mapping and sequencing of individualpeptides, respectively. Peptide mixtures subjected to MS provided ionchromatograms (FIG. 3) that were used to compare the degree of ionoverlap between the three examined molecules. Extensive ion overlapindicates that the peptides bound by these tailed and untailed B*1501molecules were nearly identical.

Selected ions were then MS/MS sequenced in order to confirm that mappedion overlaps indeed represented exact ligand matches through comparisonof fragmentation patterns between the three molecules (FIG. 4).Approximately 60 peptides were chosen initially for MS/MS—ten from eachfraction. Overall, fragmentation patterns were exact matches in amajority of the peptides examined (TABLE VI). Fragmentation patternscategorized as nonmatches resulted from a mixture of peptides present atthe same mass to charge ratio, one or more of which was present in thetagged molecule and not apparent in the spectra of the same ion fromB*1501. Of the sequence-level matches, ligands derived from HIS-tailedmolecules more closely matched those derived from B*1501 than thoseeluted from FLAG-tailed molecules. In total, 52/57 HIS peptides wereexact matches, whereas 39/56 FLAG peptides were equivalent. Thus, thedata indicates that the 6-HIS tag is less disruptive to endogenouspeptide binding than is the FLAG-tag, although neither tag drasticallyaltered the peptides bound by B*1501.

A handful of individual ligand sequences present in fractions ofpeptides eluted from all three molecules were determined by MS/MS. Thetwo clearest sequences, AVRDISEASVF and QGLISRGYSY, demonstrate thattailed class I molecules indeed load endogenous peptide ligands. Thissupports the hypothesis that addition of a C-terminal tag does notabrogate the ability of the soluble HLA-B*1501 molecule to naturallybind endogenous peptides. Further, both peptide sequences closelymatched those previously reported for B*1501 eluted peptides having adisparate N-terminus paired with a more conserved C-terminus consistingof either a phenylalanine or a tyrosine. Given the homologous Edmansequence, largely identical fragmentation patterns, and the peptideligands shared between the three molecules, we conclude that addition ofa C-terminal tag does not significantly alter the peptides bound byB*1501.

Mapping and subtractively comparing eluted peptides is a direct meansfor identifying differences and similarities in the individual ligandsbound by a class I HLA molecule. Indeed, subtractive comparisonsdemonstrate how overlapping ligands bind across closely related HLA-B15subtypes as well as pointing out which ligands are unique tovirus-infected cells. Direct comparative analyses of eluted peptideligands is well suited for a number of purposes, not the least of whichis viral and cancer CTL epitope discovery. Addition of a C-terminalepitope tag provides a feasible method for production and purificationof class I molecules, and therefore, peptide ligands in cell linescapable of sustaining viral infection or harboring neoplastic agents, asillustrated in FIG. 5. Direct peptide analysis from such lines shouldyield important information on host control of pathogenic elements aswell as provide important building blocks for rational vaccinedevelopment.

The present invention further relates in particular to a novel methodfor detecting those peptide epitopes which distinguish theinfected/tumor cell from the uninfected/non-tumor cell. The resultsobtained from the present inventive methodology cannot be predicted orascertained indirectly; only with a direct epitope discovery method canthe unique epitopes described herein be identified. Furthermore, onlywith this direct approach can it be ascertained that the source proteinis degraded into potentially immunogenic peptide epitopes. Finally, thisunique approach provides a glimpse of which proteins are uniquely up anddown regulated in infected/tumor cells.

The utility of such HLA-presented peptide epitopes which mark theinfected/tumor cell are three-fold. First, diagnostics designed todetect a disease state (i.e., infection or cancer) can use epitopesunique to infected/tumor cells to ascertain the presence/absence of atumor/virus. Second, epitopes unique to infected/tumor cells representvaccine candidates. Here, we describe epitopes which arise on thesurface of cells infected with HIV. Such epitopes could not be predictedwithout natural virus infection and direct epitope discovery. Theepitopes detected are derived from proteins unique to virus infected andtumor cells. These epitopes can be used for virus/tumor vaccinedevelopment and virus/tumor diagnostics. Third, the process indicatesthat particular proteins unique to virus infected cells are found incompartments of the host cell they would otherwise not be found in.Thus, we identify uniquely upregulated or trafficked host proteins fordrug targeting to kill infected cells.

The present invention describes, in particular, peptide epitopes uniqueto HIV infected cells. Peptide epitopes unique to the HLA molecules ofHIV infected cells were identified by direct comparison to HLA peptideepitopes from uninfected cells.

As such, and only by example, the present method is shown to be capableof identifying: (1) HLA presented peptide epitopes, derived fromintracellular host proteins, that are unique to infected cells but notfound on uninfected cells, and (2) that the intracellularsource-proteins of the peptides are uniquely expressed/processed in HIVinfected cells such that peptide fragments of the proteins can bepresented by HLA on infected cells but not on uninfected cells.

The method of the present invention also, therefore, describes theunique expression of proteins in infected cells or, alternatively, theunique trafficking and processing of normally expressed host proteinssuch that peptide fragments thereof are presented by HLA molecules oninfected cells. These HLA presented peptide fragments of intracellularproteins represent powerful alternatives for diagnosing virus infectedcells and for targeting infected cells for destruction (i.e., vaccinedevelopment).

A group of the host source-proteins for HLA presented peptide epitopesunique to HIV infected cells represent source-proteins that are uniquelyexpressed in cancerous cells. For example, through using the methodologyof the present invention a peptide fragment of reticulocalbin isuniquely found on HIV infected cells. A literature search indicates thatthe reticulocalbin gene is uniquely upregulated in cancer cells (breastcancer, liver cancer, colorectal cancer). Thus, the HLA presentedpeptide fragment of reticulocalbin which distinguishes HIV infectedcells from uninfected cells can be inferred to also differentiate tumorcells from healthy non-tumor cells. Thus, HLA presented peptidefragments of host genes and gene products that distinguish the tumorcell and virus infected cell from healthy cells have been directlyidentified. The epitope discovery method of the present invention isalso capable of identifiying host proteins that are uniquely expressedor uniquely processed on virus infected or tumor cells. HLA presentedpeptide fragments of such uniquely expressed or uniquely processedproteins can be used as vaccine epitopes and as diagnostic tools.

The methodology to target and detect virus infected cells may not be totarget the virus-derived peptides. Rather, the methodology of thepresent invention indicates that the way to distinguish infected cellsfrom healthy cells is through alterations in host encoded proteinexpression and processing. This is true for cancer as well as for virusinfected cells. The methodology according to the present inventionresults in data which indicates without reservation thatproteins/peptides distinguish virus/tumor cells from healthy cells.

Example of Comparative Ligand Mapping in Infected and Uninfected CellsCreation of Soluble Class I Construct

EBV-transformed cell lines expressing alleles of interest (particularlyA*0201, B*0702, and Cw*0702) were grown and class I HLA typed throughthe sequenced-based-typing methodology described in Turner et al. 1998,J. Immunol, 161 (3) 1406-13) and U.S. Pat. No. 6,287,764 Hildebrand etal. both of which are expressly incorporated herein in their entirety byreference. Total RNA was 5pXI and 3pEI, producing a product lacking thecytoplasmic and transmembrane domains. Alternatively, a 3′ primerencoding a hexa-histidine or FLAG epitope tag was placed on theC-terminus using the primers, 3pEIHIS or 3pEIFLAG (TABLE V). For theC-locus, a 5′ primer was used encoding the Kozak consensus sequence.(Davis, et al. 1999. J. Exp. Med. 189: 1265-1274). Each construct wascut with the appropriate restriction endonculease (see TABLE V) andcloned into the mammalian expression vector pcDNA 3.1- (Invitrogen,Carlsbad, Calif.) encoding either a resistance gene for G418 sulfate orZeocin (Invitrogen).

Transfection in Sup-T1 cells. Sup-T1 T cells were cultured in RPMI1640+20% fetal calf serum at 37° C. and 5% CO₂. Cells were split dailyin order to maintain log-phase growth. Plasmid DNA was purified usingeither Qiagen Midi-prep kits (Qiagen, Santa Clarita) or Biorad QuantumPrep Midiprep Kit (Biorad, Hercules, Calif.) according to themanufacturer's protocol and resuspended in sterile DNAse-free water.Cells were electroporated with 30 ìgs of plasmid DNA at a voltage of 400mV and a capacitance of 960 ìF. Decay constants were monitoredthroughout electroporation and only transfections with decay times under25 mS were carried through to selection. Selection was performed on day4 post-transfection with 0.45 mg/mL Zeocin (Invitrogen) selective mediumcontaining 30% fetal calf with the pH adjusted visually to just higherthan neutral. Cells were resuspended in selective medium at 2×10̂6 cellsper ml, fed until they no longer turned the wells yellow (using the pHindicated Phenol Red (Mediatech)), and allowed to sit until cells beganto divide. After the appearance of active division, cells were slowlyfed with selective medium until they reached medium (T-75) tissueculture flasks. Cells were then subcloned at limiting dilutions of 0.5,1, and 1.5 cells per well in 96-well tissue culture plates. Cells wereallowed to sit until well turned yellow; they were then gradually movedto 24 well plates and small (T-25) tissue culture flasks. Samples weretaken for soluble class I ELISA, and the best producers of class I werefrozen for later use at 5×10̂6 cells/ml and stored at −135° C.

Soluble MHC class I ELISA. ELISAs were employed to test theconcentration of the MHC class I/peptide complexes in cell culturesupernatants. The monoclonal antibody W6/32 (ATCC, Manassas, Va.) wasused to coat 96-well Nunc Starwell Maxi-sorp plates (VWR, West Chester,Pa.). One hundred ìls of test sample containing class I was loaded intoeach well of the plate. Detection was with anti-âB2 microglobulin (lightchain) antibody conjugated to horse-radish peroxidase followed byincubation with OPD (Sigma, St. Louis, Mo.). ELISA values were read by aSpectraMax 340 00A, Rom Version 2.04, February 1996, using the programSoftmax Pro Version 2.2.1 from Molecular Devices. For determination ofMHC class I complex in carboys prior to affinity purification (seebelow), each sample was tested in triplicate on at least 2 separateplates. Uninfected and infected harvest concentrations were read on thesame plate and uninfected samples were brought to 1% Triton X 100 priorto loading on the ELISA plate. This was in an attempt to minimizevariability in mass spectra generate due to large differences in theamount of peptide loaded onto affinity columns.

Full-length construct creation. Full-length constructs (in thepcDNA3.1−/G418 sulfate resistance vector) were created and transfectedinto the class I negative B-LCL 721.221 and T2. Both cell lines werecultured in RPMI-1640+10% fetal calf serum until growing at log phase.Cells were electroporated at 0.25 V and 960 ìF capacitance. After 2days, the cells were pelleted and resuspended in selective mediumconsisting of RPMI-1640+20% FCS+1.5 mg/ml G418 sulfate (Mediatech,Herndon, Va.). Cells were treated in the same manner as above (Sup-T1transfection) after this point.

Cell pharm production. Eight liters of Sup-T1 soluble MHC class Itransfectants cultured in roller bottles in RPMI-1640+15% FCS+100 Upenicillin/streptomycin were centrifuged for 10 min at 1100×g.Supernatant was discarded and a total of 3×10̂9 total cells wereresuspended in 200 mls of conditioned medium. Infected cells were thenadded to a feed bottle and inoculated through the ECS feed pump of aUnisyn CP2500 cell pharm (Unisyn, Hopkington, Mass.) into 30 kDmolecular-weight cut-off hollow-fiber bioreactors previously primed withRPMI-1640 containing 20% fetal calf serum. Cells were allowed toincubate overnight in the bioreactor at a temperature of 37° C. and at apH of 7.20 maintained automatically through CO₂ injection into themedium reservoir of the system. No new medium was introduced into thesystem during this time period and the ICS recirculation was maintainedat a low value of 400 mls/minutes. ECS feed was begun 12 hours postinoculation at a rate of 100 mls/day with 15% FCS supplementedRPMI-1640; ICS feed was likewise begun at a rate of 1 L/day. ECSrecirculation was initiated at day 2 post-inoculation at a rate of 4L/day. ECS and ICS samples were taken at 24-hour intervals and sHLAELISAs (see above) and glucose tests performed. ECS and ICS feed ratesas well as ECS and ICS recirculation rates were adjusted based onincreasing concentrations of sHLA in the harvest and decreasing levelsof glucose in the ICS medium.

Virus production and infection HIV MN-1 production. HIV MN-1 clonedvirus (Genbank Accession Number M17449) was thawed from frozen stock andused to infect 25×10̂6 non-transfected Sup-T1 (Denny C T, et. al. 1986.Nature. 320:549.51, which is expressly incorporated herein in itsentirety by reference) T cells using standard methods. Cells werecultured in RPMI-1640+20% fetal bovine serum (MediaTech) for 5 days andobserved for syncitia formation. Upon formation of syncitia, new cellswere added in fresh RPMI-1640/20% FCS. Culture was continued for 5 moredays when 100 mls of infected cells were removed. Supernatant was passedthrough a 0.45 um filter and cell-free virus was aliquotted and storedat −80° C. This process was continued until an appropriate amount ofvirus was harvested.

[ ] HIV-1 NL4-3 production. The infectious molecular clone pNL4-3(Genbank Accession Number AF324493) was transformed into the Esherichiacoli strain Top10F′ (Invitrogen, Carlsbad, Calif.). Plasmid DNA wasmidiprepped from transformed cells using either the Qiagen Midi Prep Kit(Qiagen, Santa Clarita, Calif.) or the Biorad Quantum Prep Midiprep Kit(Biorad, Hercules, Calif.) according to the manufacturer's instructions.Plasmid DNA was used to transfect 293T cells (GenHunter Corporation,Nashville, Tenn.) using Roche's Fugene 6 reagent (Roche, Basel,Switzerland) following the manufacturer's protocol. Virus-containingsupernatant was harvested at 24, 48, and 72 hours, clarified bycentrifugation at 500×g for 10 min, aliquotted, and stored at −80° C.Sup-T1 transfectants containing either soluble A*0201, B*0702, orCw*0702 were cocultured with virus resulting in high-titre virus. After72 hours, infected cells were centrifuged at 1100×g for 10 minutes.Supernatant containing cell-free virus was removed, passed through a0.45 ìm filter, aliquotted, and stored at −80° C. Virally-infected cellswere resuspended in freeze medium (RPMI-1640+20% FCS+10% DMSO) atapproximately 6×10̂6 cells per ml and stored at −80° C.

Viral Titer Determination. One vial of frozen viral stock derived fromeither strain of HIV was thawed and used in a TCID₅₀ assay scored twoways: 1) wells containing at least 3 syncitia were considered positiveor 2) wells containing over 50 ng/ml p24 antigen as determined by ELISAwere considered positive. The TCID₅₀ was then calculated using theSpearman-Karber method (DAIDS Virology Manual for HIV Laboratories,January 1997). The average of both scoring methods was used as the finaltiter of the virus. As a second means of viral titer monitoring, viralstock was used undiluted in a p24 ELISA (Beckman Coulter, Miami, Fla.)in order to determine the ngs of p24 present in cell-free virus.

P24 ELISA. Determination of HIV p24 major core protein was determined bythe commercially available Beckman Coulter p24 ELISA according to themanufacturer's instructions with the exceptions of the followingmodifications: samples were treated with 10% Triton-X 100 prior toremoval from a BSL-3 facility, therefore the inactivation mediumincluded in the kit was not used. Secondly, samples were seriallydiluted in water prior to use.

Hollow-fiber bioreactor culture of infected cells. All work includinglarge-scale culture of HIV was performed in a Biosafety Level 3Laboratory in accordance with guidelines set forth by the NationalInstitutes of Health. HIV MN-1 frozen viral stock aliquots were thawedand pooled to a 100 ml total volume, containing approximately 5.5×10̂6TCID₅₀'s. Eight liters of Sup-T1 soluble MHC class I transfectantscultured in roller bottles in RPMI-1640+15% FCS+100 Upenicillin/streptomycin were centrifuged for 10 min at 1100×g.Supernatant was discarded and a total of 3×10̂9 total cells wereresuspended in 200 mls of conditioned medium. The 100 mls of cell-freeHIV MN-1 was then added to the resuspended cells and incubated at 37° C.in %5 CO₂ for 2 hours with gentle shaking every 20 minutes. Infectedcells were then added to a feed bottle and inoculated through the ECSfeed pump of a Unisyn CP2500 cell pharm (Unisyn, Hopkington, Mass.) into30 kD molecular-weight cut-off hollow-fiber bioreactors previouslyprimed with RPMI-1640 containing 20% fetal calf serum. Cells wereallowed to incubate overnight in the bioreactor at a temperature of 37°C. and at a pH of 7.20 maintained automatically through CO₂ injectioninto the medium reservoir of the system. No new medium was introducedinto the system during this time period and the recirculation wasmaintained at a low value of 400 mls/minutes. ECS feed was begun 12hours post inoculation at a rate of 100 mls/day with 15% FCSsupplemented RPMI-1640; ICS feed was likewise begun at a rate of 1L/day. ECS and ICS samples were taken at 24-hour intervals, inactivatedby addition of Triton-X 100 to 1%, and sHLA ELISAs, p24 ELISAs, andglucose tests performed as described above. ECS and ICS feed rates aswell as ECS and ICS recirculation rates were adjusted based onincreasing concentrations of sHLA in the harvest and decreasing levelsof glucose in the ICS medium.

Soluble HLA purification. Soluble-HLA containing supernatant was removedin 1.9 L volumes from infected hollow-fiber bioreactors. Twenty-percentTriton-X 100 was sterilized and placed in 50 ml aliquots in 60 mlssyringes; 2 syringes were injected into each 1.9 L harvest bottle as itwas removed from the cell pharm, resulting in a final TX 100 percentageof 1%. Bottles were inverted gently several times to mix the TX 100 andstored at 4° C. for a minimum of 1 week. After 1 week, harvest wascentrifuged at 2000×g for 10 minutes to remove cellular debris andpooled into 10 L carboys. An aliquot was then removed from the pooled,HIV-inactivated supernatant and used in a quantitative TCID₅₀ assay (asdescribed above) and used to initiate a coculture with Sup-T1's. Onlyafter demonstration of a completely negative coculture as well as TCID₅₀were harvests removed from the BSL-3.

Class I/Peptide Production and Peptide Characterization Handling of MHCclass I/peptide complexes from infected cells. Each 10 L of cell pharmharvest was separated strictly on a temporal basis during the cell pharmrun. (This was an attempt to assess any epitopic changes that mightoccur temporally during infection as opposed to those that might occurmore globally.) Harvest was treated exactly as described above, exceptfor the removal of a 2 ml aliquot for tests in both a TCID₅₀ assay andcell coculture assay to determine infectivity of the virus.

Affinity purification of infected and uninfected MHC class I complexes.Uninfected and infected harvest removed from CP2500 machines weretreated in an identical manner post-removal from the cell pharm.Approximately 50 mgs total class I as measured by W6/32 ELISA (seeabove) were passed over a Pharmacia XK-50 (Amersham-Pharmacia Biotech,Piscataway, N.J.) column packed with 50 mls Sepharose Fast Flow 4Bmatrix (Amersham) coupled to W6/32 antibody. Bound class I complexeswere washed first with 1 L 20 mM sodium phosphate wash buffer, followedby a wash with buffer containing the zwitterionic detergent Zwittergent3-08 (Calbiochem, Merck KgaA, Darmstadt, Germany) at a concentration of10 mM, plus NaCl at 50 mM, and 20 mM sodium phosphate. The zwittergentwash was monitored by UV absorption at a wavelength of 216 nm forremoval of Triton-X 100 hydrophobically bound to the peptide complexes.After 1 L of wash had passed over the column (more than a sufficientamount for the UV to return to baseline), zwittergent buffer was removedwith 2 L of 20 mM sodium phosphate wash buffer. Peptides were elutedpost wash with freshly made 0.2N acetic acid, pH 2.7.

Peptide isolation and separation. Post-elution, peptide-containingeluate fractions were brought up to 10% glacial acetic acidconcentration through addition of 100% glacial acetic acid. Fractionswere then pooled into a model 8050 stirred cell (Millipore, Bedford,Mass.) ultrafiltration device containing a 3 kD molecular-weight cutoffregenerated cellulose membrane (Millipore). The device was capped andtubing parafilmed to prevent leaks and placed in a 78° C. water bath for10 minutes. Post-removal, the peptide-containing elution buffer wasallowed to cool to room temperature. The stirred cell was operated at apressure of 55 psi under nitrogen flow. Peptides were collected in 50 mlconical centrifuge tubes (VWR, West Chester, Pa.), flash frozen insuper-cooled ethanol, and lyophilized to dryness. Peptides wereresuspended either in 10% acetic acid or 10% acetonitrile. Peptides werepurified through a first-round of HPLC on a Haisil C-18 column (HigginsAnalytical, Moutain View, Calif.), with an isocratic flow of 100% B(100% acetonitrile, 0.01% TFA) for 40 minutes. Following elution,peptide-containing fractions were pooled, speed-vacuumed to dryness, andresuspended in 150 ìls of 10% acetic acid. Two igs of the base methylviolet were added to the peptide mixture in 10% acetic acid and this wasloaded onto a Haisil C-18 column for fractionation. Peptides werefractionated by one of two methods, the latter resulting in increasedpeptide resolution. The first fractionation program was 2-10% B in 2minutes, 10-60% B in 60 minutes, with 1 minute fraction collection. Thesecond RP-HPLC gradient consisted of a 2-14% B in 2 minutes, 14-40% B in60 minutes, 40-70% B in 20 minutes, with 1 minute fraction collection.Peptides eluting in a given fraction were monitored by UV absorbance at216 nm. Separate but identical (down to the same buffer preparations)peptide purifications were done for each peptide-batch from uninfectedand infected cells.

Mass-spectrometric mapping of fractionated peptides. Fractionatedpeptides were mapped by mass spectrometry to generate fraction-based ionmaps. Fractions were speed-vacuumed to dryness and resuspended in 12 ìls50:50 methanol:water+0.05% acetic acid. Two ìls were removed and sprayedvia nanoelectrospray (Protana, Odense, Denmark) into a Q-Star quadrupolemass spectrometer with a time-of-flight detector (Perseptive SCIEX,Foster City, Calif.). Spectra were generated for masses in the range of50-1200 amu using identical mass spectrometer settings for each fractionsprayed. Spectra were then base-line subtracted and analyzed using theprograms BioMultiview version 1.5beta9 (Persceptive SCIEX) or BioAnalystversion 1.0 (Persceptive SCIEX). Spectra from the same fraction inuninfected/infected cells were manually aligned to the same mass range,locked, and 15 amu increments visually assessed for the presence ofdifferences in the ions represented by the spectra (for an example, seeHickman et al. 2000. Human Immunology. 61:1339-1346 which is expresslyincorporated herein by reference). Ions were selected for MS/MSsequencing based on upregulations or downregulation of 1.5 fold over thesame ion in the uninfected cells, or the presence or absence of the ionin infected cells. Ions were thus categorized into multiple categoriesprior to MS/MS sequencing.

Tandem mass-spectrometric analysis of selected peptides. Lists of ionsmasses corresponding to each of the following categories weregenerated: 1) upregulated in infected cells, 2) downregulated ininfected cells, 3) present only in infected cells, 4) absent in infectedcells, and 5) no change in infected cells. The last category wasgenerally disregarded for MS/MS analysis and the first 4 categories weresubjected to MS/MS sequencing on the O-Star mass spectrometer.Peptide-containing fractions were sprayed into the mass spectrometer in3 ìl aliquots. All MS settings were kept constant except for the Q0 andCad gas settings, which were varied to achieve the best fragmentation.Fragmentation patterns generated were interpreted manually and with theaid of BioMultiView version 1.5 beta 9. No sequencing algorithms wereused for interpretation of data, however multiple web-based applicationswere employed to aid in peptide identification including: MASCOT(Perkins, D N et al. 1999. Electrophoresis. 20(18):3551-3567), ProteinProspector (Clauser K. R. et al. 1999. Analytical Chemistry. 71:2871),PeptideSearch(narrador.emblheidelberg.de/GroupPages/PageLink/Peptidesearchpage.html)and BLAST search (www.ncbi.nlm.nih.gov/BLAST/).

Quality control of epitope changes. Multiple parameters were establishedbefore peptides identified in the above fashion were deemed“upregulated,” “downregulated,” etc. First, the peptide fractions beforeand after the fraction in which the peptide was identified weresubjected to MS/MS at the same amu under the identical collisionconditions employed in fragmentation of the peptide-of-interest and thespectra generated overlaid and compared. This was done to make surethat, in the unlikely event that the peptides had fractionateddifferently (even with methyl-violet base B standardization) there wasnot the presence of the peptide in an earlier or later fraction of theuninfected or infected peptides (and that the peptides had trulyfractionated in an identical manner.) Secondly, the same amu that wasused to identify the first peptide was then subjected to MS/MS in thealternate fraction (either infected or uninfected, whichever wasopposite of the fraction in which the peptide was identified.) Spectraagain were overlaid in order to prove conclusively that thefragmentation patterns did not match and thus the peptide was notpresent in the uninfected cells, or, in the case that the fragmentationpatterns did match, that the peptides were upregulated in the infectedcells. Finally, synthetic peptides were generated for each peptideidentified. These peptides were resuspended in 10% acetic acid andRP-HPLC fractionated under the same conditions as employed for theoriginal fractionation, ensuring that the peptide putatively identifiedhad the same hydrophobicity as that of the ion MS/MS fragmented. Thissynthetic peptide was MS/MS fragmented under the same collisionconditions as that of the ion, the spectra overlaid, and checked for anexact match with the original peptide fragment.

Functional Analysis\Literature Searches. After identification ofepitopes, literature searches were performed on source proteins todetermine their function within the infected cell. Broad inferences canbe made from the function of the protein. Source proteins wereclassified into groups according to functions inside the cell. Again,broad inferences can be made as to the groups of proteins that would beavailable for specific presentation solely on infected cells. Secondly,source proteins were scanned for other possible epitopes which may bebound by other MHC class I alleles. Peptide binding predictions (Parker,K. C., et. al. 1994. J. Immunol. 152:163) were employed to determine ifother peptides presented from the source proteins were predicted tobind. Proteasomal prediction algorithms (A. K. Nussbaum, et. al. 2001.Immunogenetics 53:87-94) were likewise employed to determine thelikelihood of a peptide being created by the proteasome.

Sequence Identification. A discussion of the results seen with theapplication of this procedure is included using the peptide GPRTAALGLLas an example. Other examples and data obtained based on the methodologyare listed in TABLE VII.

TABLE VII ION FRACTION SEQUENCE MW OBS′D MWPeptides Identified on Infected cells that are not present on Uninfected Cells612.720 32INF EQMFEDIISL 1223.582 1223.418 509.680 31INF IPCLLISFL1017.601 1017.334 469.180 31INF STTAICATGL 936.466 936.360 420.130 16INFAPAQNPEL 838.426 838.259 500.190 28INF LVMAPRTVL 998.602 998.396 529.68031INF APFI[NS]PADX 1057.388 523.166 12INF TPQSNRPVm 1044.500 1044.333444.140 16INF AARPATSTL 887.495 887.280 470.650 16INF MAMMAALMA 940.413939.410 490.620 16INF IATVDSYVI 979.240 563.640 16INF SPNQARAQAAL1126.597 1126.364 30INF GPRTAALGLL 968.589 968.426 556.150 16INFNPNQNKNVAL 1111.586 1111.300Peptides Identified on Uninfected cells that are not present on Infected cells16UNINF GSHSMRY ION SOURCE PROTEIN START AA ACCESSION #Peptides Identified on Infected cells that are not present on Uninfected Cells612.720 HIV MN-1, ENV 101 509.680 CHOLINERGIC RECEPTOR, 250ALPHA-3 POLYPEPTIDE 469.180 UBIQUITIN-SPECIFIC PROTEASE 152 10720340(SEQ ID NO: 43) 420.130 B-ASSOCIATED TRANSCRIPT PROTEIN 3 (BAT3) 500.190HLA-B HEAVY CHAIN LEADER SEQUENCE 2 4566550 (SEQ ID NO: 44) 529.680UNKNOWN, CLOSE TO SEVERAL cDNA′s 523.166 RNA POLYMERASE II POLYPEPTIDE A527 4505939 (SEQ ID NO: 45) 444.140 EUK, TRANSLATION INITIATION FACTOR 41073 Q04637 (SEQ ID NO: 46) 470.650 SPARC-LIKE PROTEIN 19 478522(SEQ ID NO: 47) 490.620 TENASCIN-C (HEXABRACHION) 1823 13639246(SEQ ID NO: 48) 563.640 POLYPYRIMIDINE TRACT-BINDING PROTEIN 1 141131528 (SEQ ID NO: 49) RETICULOCALBIN 4 4506457 (SEQ ID NO: 50) 556.150ELAV (HuR) 188 4503551 (SEQ ID NO: 51)Peptides Identified on Uninfected cells that are not present on Infected cellsMHC CLASS I HEAVY CHAIN variable multiple(could derive from multiple alleles, i.e., HLA-B*0702 or HLA-G, etc.)ION CATEGORY SEQ ID NO:Peptides Identified on Infected cells that are not present on Uninfected Cells612.720 HIV-DERIVED 29 509.680 30 469.180 31 420.130 MHC GENE PRODUCT 32500.190 MHC GENE PRODUCT 33 529.680 UNKNOWN 34 523.166 RNA MACHINERY/ 35BINDING PR 444.140 RNA MACHINERY/ 36 BINDING PR 470.650 TUMOR SUPPRESSOR37 GENE? 490.620 TUMOR SUPPRESSOR 38 GENE? 563.640 RNA MACHINERY/ 39BINDING PR TUMOR SUPPRESSOR 40 GENE? 556.150 RNA MACHINERY/ 41BINDING PRPeptides Identified on Uninfected cells that are not present on Infected cellsMHC CLASS I 42 Product

The first step in identification of an epitope present only onuninfected cells is performing MS ion mapping. In this case, thereversed-phase HPLC fraction 30 obtained from HIV as disclosedhereinabove (which contains a fraction of the total class I peptides)was sprayed into the mass spectrometer and an ion spectrum created. FIG.6 shows the sections of ion map in which an ion was first identified asupregulated. The ion at 484.74 can be seen to predominate in the uppermap, which is the spectrum generated from peptides from the infectedcells. One can also see that there are other peptides which differ intheir intensities between the uninfected cells from one spectrum toanother. After a peptide is initially identified, the area of thespectrum in which the peptide is found is zoomed in on in order to morefully see all the ions in the immediate area (FIG. 7). After zooming inon the area from 482-488 amu, the ion at 484.72 can be seen to only bepresent in the infected cells (which are seen in the spectrum on thetop). A large difference such as this is not always seen, sometimes moreminor differences are chosen for sequence determination. This ion,however, was considered an extremely good candidate for furtheranalysis.

After identification of the ion, the next step in the process is tosequence the peptide by using tandem mass spectrometry. FIG. 8 shows thespectrum generated when the peptide is fragmented. These fragments areused to discern the amino acid sequence of the peptide. The sequence ofthis peptide was determined to be GPRTAALGLL. This peptide was isolatedfrom infected HLA-B*0702 molecules. One early quality control step isexamining the peptide's sequence to see if it fits the sequences thatwere previously shown to be presented by this molecule. B*0702 bindspeptides that have a G at their second position (P2) and an L as theirC-terminal anchor. Based on this information, this sequence is likely tobe a peptide presented by B*0702.

Descriptive characterization of peptide. Once the peptide sequence isobtained, information is gained on the source protein from which thepeptide was derived in the cytosol of the infected cell. Initially, aBLAST search (available at the National Center for Biotechnologywebsite) is done to provide protein information on the peptide. A BLASTsearch with the sequence GPRTAALGLL pulled up the protein reticulocalbin2. After the source protein is known, information about the protein isascertained first from the PubMed (again available at the NationalCenter for Biotechnology website) and put into a format to which one caneasily refer as seen in FIG. 9. All of the accession numbers for theprotein, as well as the original description of the protein areincluded. This makes it easy to come back to the information fordownstream use. Also, the protein sequence is copied, pasted, and savedas a text document for incorporation into later searches. The peptide ishighlighted in the entire protein, giving some context as to where it isderived and how large the total protein is. This is the initial datagathering step post-sequence determination.

The next step in characterizing the ligand is doing literature searcheson the source protein from which the peptide was derived. The protein isentered into the PubMed database and all entries with the word“reticulocalbin” are retrieved. FIG. 10 illustrates the listing that isdone to summarize what has previously been described for this protein.It can be seen that for reticulocalbin, multiple articles have beenpublished involving this protein. The literature is summarized in aparagraph following the PubMed listings and put into the report. Forreticulocalbin, some of the most interesting points are that it is an ERresident protein, which can lead to speculation on why it is presentedon infected cells. Secondly, it has been previously found to beupregulated in several other types of cancers, such as breast andcolorectal cancers. This again leads to speculation that this proteinmay be broadly applicable to treat more maladies than those caused byHIV. It is also determined whether or not this protein has beenpreviously cited as interacting with/or being interfered with by HIV.This was not seen for reticulocalbin and thus was not listed in thereport, (although in some instances it is seen.) A broad understandingof the protein is gained through literature searches.

Predictive characterization of peptide. After the literature search,several secondary searches are performed. FIG. 11 illustrates theresults of a peptide-binding algorithm performed using Parker'sPrediction (which is described hereinabove). The entire source proteinis used for input and the computer generates a list of peptides whichare bound by the HLA allele chosen. In this case, B*0702 was chosenbecause that was the allele from which this peptide was derived. Fromthe black arrow in the figure, it can be seen that the peptide sequencedby mass spectrometry is predicted to bind to HLA-B*0702 with a highaffinity. Several other peptides are listed that are predicted to bindas well. FIG. 12 shows the same procedure being performed with thesource peptide using another well-known search engine, SYPEITHI. (Thisengine can be found on the worldwide web using the URL:syfpeithi.bmi-heidelberg.com/Scripts/MHCServer.dll/EpPredict.htm.)Again, the results from this search engine for B*0702 shows that thispeptide is predicted to bind to HLA-B*0702 with a high affinity. Also,multiple other peptides are predicted to be derived from this sourceprotein and bound. This prediction allows us to determine severalthings. First, we can tell if the peptide is predicted to be bound byprevious algorithms. This allows us to know how well the programs work,and/or if other people could identify this peptide (if they had thesource protein) from peptide binding algorithms. All of this informationcan be translated into increasing importance for the present inventivemethodology not only for the peptide but also for the source proteinitself.

After peptide-binding algorithms are performed, searches are done todetermine whether the peptides would be created by the proteasome duringnormal processing of proteins into peptides. It should be strongly notedthat multiple pathways for class I peptide loading are now beingdemonstrated and that the cleavage algorithms for human proteasomes arenot well established by any means. While a positive result may indicatethat the proteasome is largely responsible for cleavage, a negativeresult by no means indicates that the peptide is not presented in theclass I molecule. FIG. 13 shows the results of the first proteasomalcleavage done for the source protein reticulocalbin using the cleavagepredictor PaProC (available at URL paproc.de/). The epitope is outlined.By this prediction software, the peptide is not predicted to be cleavedby the normal proteasome. This may mean that in infected cells,alternative pathways of MHC class I presentation are being used,particularly in reference to the reticulocalbin peptide. This, in turn,may present novel methods for therapeutics during viral infection. Asecond proteasomal cleavage search is also employed using the predictionsoftware NetChop (available on the worldwide web) as seen in FIG. 14. Bythis prediction and other data from current literature in the field, thepeptide would be created by the proteasome and cleaved to form theGPRTAALGLL identified.

A third round of analysis involves only the source protein. All otheralleles are tested for peptide binding and lists of the highest bindersgenerated. The proteasomal cleavage predictions are then referred to inorder to elucidate how these peptides are generated. This information isuseful for downstream testing of peptides and for determining whether ornot this protein will be applicable for vaccine trials covering a broadrange of HLA alleles. For reticulocalbin, multiple high-affinitypeptides were demonstrated for differing HLA alleles (some examples ofwhich are shown in FIG. 15) In this figure, several high affinitypeptides deriving from reticulocalbin were identified for HLA-A*0201 andA*0101.

Quality control of sequence determination. There currently exists nodirect means to score the quality of MS/MS sequence data. Once alldescriptive and predictive steps are concluded, we return again to theoriginal peptide sequence for quality control to ensure that the peptideis indeed what we have identified as the amino acid sequence and thatthe peptide is truly present only in infected cells. We employ thesemultiple steps so there is no doubt that the sequence is truly what weclaim it to be before we move on to downstream applications involvingthe peptide.

Initially, we determine that the peptide is truly upregulated or presentonly in infected cells. For the reticulocalbin peptide, we determinedthat this peptide was probably only present in infected cells. In orderto make certain that the peptide was truly absent in the uninfectedcells and that there was no chance that our RP-HPLC fractionation haddiffered (remembering that we use internal controls for ourfractionation as well) we generated ion spectra using MS from thefractions before and after the one in which we identified the peptide.In the case of the reticulocalbin peptide, we identified the peptide infraction 30, so we performed MS on fractions 29 and 31 (FIG. 16). InFIG. 16, it can be seen that there is no substantial peak at the m/z484.72. This indicated that there was not differential fractionation andthat the peptide truly was absent from uninfected cells. In the casethat there was a peptide peak in one of the before or after fractions,we would then turn to MS/MS to determine whether this peak representedthe ion we were characterizing or another ion with the samemass-to-charge ratio.

After determining that the peptide is not present in another fraction,MS/MS was preformed on the same m/z in the uninfected spectrum (in thesame fraction) in order to conclusively prove that there is no peptidepresent with the same sequence in the uninfected cells. In FIG. 17 onecan see that the fragmentation patterns produced under identical MScollision conditions are totally different. This illustrates the absenceof the reticulocalbin peptide in the uninfected cells.

Finally, in order to conclusively prove that the peptide sequence is thesame as that originally identified, we synthesize synthetic peptidesconsisting of the same amino acids as the peptide sequence identifiedfrom the MS/MS fragmentation pattern. For the reticulocalbin peptide(i.e., the ion in fraction 30 at 484.72) we synthesized the peptide“GPRTAALGLL.” We then took this peptide and did MS/MS on the peptideunder identical conditions as previously used. FIG. 18 illustrates thespectrum generated from MS/MS of the endogenously loaded reticulocalbinpeptide. Matching spectra, as seen here, are indicators that thispeptide sequence is GPRTAALGLL as almost every amino acid combinationwill generate a completely different set of fragments, both in terms ofproduction of fragments and in terms of intensity of those fragmentspresent. FIG. 18 shows the MS/MS endogenous and synthetic “GPRTAALGLL”peptide under identical collision conditions. As can be seen, the MS/MSgraphs are virtually identical.

In accordance with the present invention, one peptide ligand (i.e.,“GPRTAALGLL”) has been identified as being presented by the B*0702 classI MHC molecule in cells infected with the HIV MN-1 virus but not inuninfected cells. As one of ordinary skill in the art can appreciate thenovelty and usefulness of the present methodology in directlyidentifying such peptide ligands and the importance such identificationhas for numerous therapeutic (vaccine development, drug targeting) anddiagnostic tools. As such, numerous other peptide ligands have beenuniquely identified in cells infected with HIV MN-1 (as opposed touninfected cells_ and these results are summarized in TABLE VII. One ofordinary skill in the art given the present specification would be fullyenabled to identify the “GPRTAALGLL” peptide ligand; as well as otheruniquely presented peptide ligands found in cells infected with amicroorganism of interest and/or tumorigenic cells.

As stated above, TABLE VII identifies the sequences of peptide ligandsidentified to date as being unique to HIV infected cells. Class I sHLAB*0702 was harvested for T cells infected and not infected with HIV.Peptide ligands were eluted from B*0702 and comparatively mapped on amass spectrometer so that ions unique to infected cells were apparent.Ions unique to infected cells (and one ligand unique to uninfectedcells) were subjected to mass spectrometric fragmentation for peptidesequencing. Column 1 indicates the ion selected for sequencing, column 2is the HPLC fraction, column 3 is the peptide sequence, column 4 is thepredicted molecular weight, column 5 is the molecular weight we found,column 6 is the source protein for the epitope sequenced, column 7 iswhere the epitope starts in the sequence of the source protein, column 8is the accession number, and column 9 is a descriptor which brieflyindicates what is known of that epitope and/or its source protein.

The methodology used herein is to use sHLA to determine what is uniqueto unhealthy cells as compared to healthy cells. Using sHLA to surveythe contents of a cell provides a look at what is unique to unhealthycells in terms of proteins that are processed into peptides. TABLE VIIshows the utility of the method described herein for discoveringepitopes and their source proteins which are unique to HIV infectedcells. A detailed description of the peptide from Reticulocalbin isprovided hereinabove. The other epitopes and corresponding sourceproteins described in TABLE VII were processed in the same manner as thereticulocalbin epitope and source protein were—i.e., as described hereinabove. The data summarized in TABLE VII shows that the epitope discoverytechnique described herein is capable of identifying sHLA bound epitopesand their corresponding source proteins which are unique toinfected/unhealthy cells.

Likewise, and as is shown in TABLE VII, peptide ligands presented inindividual class I MHC molecules in an uninfected cell that are notpresented by individual class I MHC molecules in an uninfected cell canalso be identified. The peptide “GSHSMRY”, for example, was identifiedby the method of the present invention as being an individual class IMHC molecule which is presented in an uninfected cell but not in aninfected cell.

The utility of this data is at least threefold. First, the dataindicates what comes out of the cell with HLA. Such data can be used totarget CTL to unhealthy cells. Second, antibodies can be targeted tospecifically recognize HLA molecules carrying the ligand described.Third, realization of the source protein can lead to therapies anddiagnostics which target the source protein. Thus, an epitope unique tounhealthy cells also indicates that the source protein is unique in theunhealthy cell.

The methods of epitope discovery and comparative ligand mappingdescribed herein are not limited to cells infected by a microorganismsuch as HIV. Unhealthy cells analyzed by the epitope discovery processdescribed herein can arise from virus infection or also canceroustransformation. In addition, the status of an unhealthy cell can also bemimicked by transfecting a particular gene known to be expressed duringviral infection or tumor formation. For example, particular genes of HIVcan be expressed in a cell line as described (Achour, A., et al., AIDSRes Hum Retroviruses, 1994. 10(1): p. 19-25; and Chiba, M., et al., CTL.Arch Virol, 1999. 144(8): p. 1469-85, all of which are expresslyincorporated herein by reference) and then the epitope discovery processperformed to identify how the expression of the transferred genemodifies epitope presentation by sHLA. In a similar fashion, genes knownto be upregulated during cancer (Smith, E. S., et al., Nat Med, 2001.7(8): p. 967-72, which is expressly incorporated herein by reference)can be transferred in cells with sHLA and epitope discovery thencompleted. Thus, epitope discovery with sHLA as described herein can becompleted on cells infected with intact pathogens, cancerous cells orcell lines, or cells into which a particular cancer, viral, or bacterialgene has been transferred. In all these instances the sHLA describedhere will provide a means for detecting what changes in terms of epitopepresentation and the source proteins for the epitopes.

Thus, in accordance with the present invention, there has been provideda methodology for epitope discovery and comparative ligand mapping whichincludes methodology for producing and manipulating Class I and Class IIMHC molecules from gDNA that fully satisfies the objectives andadvantages set forth herein above. Although the invention has beendescribed in conjunction with the specific drawings, experimentation,results and language set forth herein above, it is evident that manyalternatives, modifications, and variations will be apparent to thoseskilled in the art. Accordingly, it is intended to embrace all suchalternatives, modifications and variations that fall within the spiritand broad scope of the invention.

1. A method for identifying at least one individual, endogenously loadedpeptide ligand for an individual class I MHC molecule that distinguishesa transfected cell from a non-transfected cell, comprising the steps of:providing a non-transfected cell line containing a construct thatencodes an individual soluble class I MHC molecule, the non-transfectedcell line being able to naturally process proteins into peptide ligandscapable of being loaded into antigen binding grooves of class I MHCmolecules; transfecting a portion of the non-transfected cell line withat least one of a gene from a microorganism and a tumor gene, therebyproviding a transfected cell line; culturing the non-transfected cellline and the transfected cell line under conditions which allow forexpression of the individual soluble class I MHC molecules from theconstruct, such conditions also allowing for endogenous loading of apeptide ligand in the antigen binding groove of each individual solubleclass I MHC molecule prior to secretion of the individual soluble classI MHC molecules from the cell; isolating the secreted individual solubleclass I MHC molecules having the endogenously loaded peptide ligandsbound thereto from the non-transfected cell line and the transfectedcell line; separating the endogenously loaded peptide ligands from theindividual soluble class I MHC molecules from the non-transfected cellline and separating the endogenously loaded peptide ligands from theindividual soluble class I MHC molecules from the transfected cell line;isolating the endogenously loaded peptide ligands from thenon-transfected cell line and the endogenously loaded peptide ligandsfrom the transfected cell line; comparing the endogenously loadedpeptide ligands isolated from the transfected cell line to theendogenously loaded peptide ligands isolated from the non-transfectedcell line; and identifying at least one individual, endogenously loadedpeptide ligand presented by the individual soluble class I MHC moleculeon the transfected cell line that is not presented by the individualsoluble class I MHC molecule on the non-transfected cell line.
 2. Themethod of claim 1, further comprising the step of identifying a sourceprotein from which the at least one individual, endogenously loadedpeptide ligand presented by the individual soluble class I MHC moleculeon the transfected cell line and not presented by the individual solubleclass I MHC molecule on the non-transfected cell line is obtained. 3.The method of claim 1 wherein, in the step of identifying at least oneindividual, endogenously loaded peptide ligand presented by theindividual soluble class I MHC molecule on the transfected cell line butnot on the non-transfected cell line, the at least one individual,endogenously loaded peptide ligand is obtained from a protein encoded byat least one of the gene from a microorganism and a tumor gene withwhich the cell line was transfected to form the transfected cell line.4. The method of claim 1 wherein, in the step of identifying at leastone individual, endogenously loaded peptide ligand presented by theindividual soluble class I MHC molecule on the transfected cell line butnot on the non-transfected cell line, the at least one individual,endogenously loaded peptide ligand is obtained from a protein encoded bythe non-transfected cell line.
 5. The method of claim 4, wherein theprotein encoded by the non-transfected cell line from which the at leastone individual, endogenously loaded peptide ligand is obtained hasincreased expression in a tumor cell line.
 6. The method of claim 1wherein, in the step of providing a non-transfected cell line containinga construct that encodes an individual soluble class I MHC molecule, theconstruct further encodes a tag which is attached to the individualsoluble class I MHC molecule and aids in isolating the individualsoluble class I MHC molecule.
 7. The method of claim 1, wherein thenon-transfected cell line is class I MHC negative.
 8. The method ofclaim 1, wherein the non-transfected cell line expresses endogenousclass I MHC molecules.
 9. The method of claim 1 wherein, in the step ofproviding a non-transfected cell line containing a construct thatencodes an individual soluble class I MHC molecule, the non-transfectedcell line containing the construct that encodes the individual solubleclass I MHC molecule is produced by a method comprising the steps of:obtaining genomic DNA or cDNA encoding at least one class I MHCmolecule; identifying an allele encoding an individual class I MHCmolecule in the genomic DNA or cDNA; PCR amplifying the allele encodingthe individual class I MHC molecule in a locus specific manner such thata PCR product produced therefrom encodes a truncated, soluble form ofthe individual class I MHC molecule; cloning the PCR product into anexpression vector, thereby forming a construct that encodes theindividual soluble class I MHC molecule; and transfecting the constructinto a non-transfected cell line.
 10. The method of claim 9 wherein, inthe step of providing a non-transfected cell line containing a constructthat encodes an individual soluble class I MHC molecule, the constructfurther encodes a tag which is attached to the individual soluble classI MHC molecule and aids in isolating the individual soluble class I MHCmolecule.
 11. The method of claim 10, wherein the tag is selected fromthe group consisting of a HIS tail and a FLAG tail.
 12. The method ofclaim 10, wherein the tag is encoded by a PCR primer utilized in thestep of PCR amplifying the allele encoding the individual class I MHCmolecule.
 13. The method of claim 11 wherein the tag is encoded by theexpression vector into which the PCR product is cloned.
 14. The methodof claim 1 wherein, in the step of transfecting a portion of thenon-transfected cell line, the portion of the non-transfected cell lineis transfected with a gene from HIV.
 15. The method of claim 1, whereinthe transfected cell is further defined as a tumorigenic cell, and thenon-transfected cell is further defined as a non-tumorigenic cell,whereby the step of transfecting a portion of the non-tumorigenic cellline with at least one of a gene from a microorganism and a tumor gene,thereby provides a transformed, tumorigenic cell line.
 16. The method ofclaim 1, further comprising the steps of: determining the source proteinfrom which the at least one individual, endogenously loaded peptideligand is obtained; and identifying the source protein as a self proteinif the source protein is not encoded by the gene from a microorganism ortumor gene with which the transfected cell line is transfected but isencoded by the non-transfected cell line.
 17. The method of claim 16,wherein the transfected cell is further defined as a tumorigenic cell,and the non-transfected cell is further defined as a non-tumorigeniccell, whereby the step of transfecting a portion of the non-tumorigeniccell line with at least one of a gene from a microorganism and a tumorgene, thereby provides a transformed, tumorigenic cell line.
 18. Amethod for identifying at least one individual, endogenously loadedpeptide ligand for an individual class I MHC molecule that distinguishesa transfected cell from a non-transfected cell, comprising the steps of:providing a non-transfected cell line containing a construct thatencodes an individual soluble class I MHC molecule, the non-transfectedcell line being able to naturally process proteins into peptide ligandscapable of being loaded into antigen binding grooves of class I MHCmolecules; transfecting a portion of the non-transfected cell line withat least one of a gene from a microorganism and a tumor gene, therebyproviding a transfected cell line; culturing the non-transfected cellline and the transfected cell line under conditions which allow forexpression of the individual soluble class I MHC molecules from theconstruct, such conditions also allowing for endogenous loading of apeptide ligand in the antigen binding groove of each individual solubleclass I MHC molecule prior to secretion of the individual soluble classI MHC molecules from the cell; isolating the secreted individual solubleclass I MHC molecules having the endogenously loaded peptide ligandsbound thereto from the non-transfected cell line and the transfectedcell line; separating the endogenously loaded peptide ligands from theindividual soluble class I MHC molecules from the non-transfected cellline and separating the endogenously loaded peptide ligands from theindividual soluble class I MHC molecules from the transfected cell line;isolating the endogenously loaded peptide ligands from thenon-transfected cell line and the endogenously loaded peptide ligandsfrom the transfected cell line; comparing the endogenously loadedpeptide ligands isolated from the non-transfected cell line to theendogenously loaded peptide ligands isolated from the transfected cellline; and identifying at least one individual, endogenously loadedpeptide ligand presented by the individual soluble class I MHC moleculeon the non-transfected cell line that is not presented by the individualsoluble class I MHC molecule on the transfected cell line.
 19. Themethod of claim 18 wherein, in the step of providing a non-transfectedcell line containing a construct that encodes an individual solubleclass I MHC molecule, the construct further encodes a tag which isattached to the individual soluble class I MHC molecule and aids inisolating the individual soluble class I MHC molecule.
 20. The method ofclaim 18, wherein the non-transfected cell line is class I MHC negative.21. The method of claim 18, wherein the non-transfected cell lineexpresses endogenous class I MHC molecules.
 22. The method of claim 18wherein, in the step of providing a non-transfected cell line containinga construct that encodes an individual soluble class I MHC molecule, thenon-transfected cell line containing the construct that encodes theindividual soluble class I MHC molecule is produced by a methodcomprising the steps of: obtaining genomic DNA or cDNA encoding at leastone class I MHC molecule; identifying an allele encoding an individualclass I MHC molecule in the genomic DNA or cDNA; PCR amplifying theallele encoding the individual class I MHC molecule in a locus specificmanner such that a PCR product produced therefrom encodes a truncated,soluble form of the individual class I MHC molecule; cloning the PCRproduct into an expression vector, thereby forming a construct thatencodes the individual soluble class I MHC molecule; and transfectingthe construct into a non-transfected cell line.
 23. The method of claim22 wherein, in the step of providing a non-transfected cell linecontaining a construct that encodes an individual soluble class I MHCmolecule, the construct further encodes a tag which is attached to theindividual soluble class I MHC molecule and aids in isolating theindividual soluble class I MHC molecule.
 24. The method of claim 23,wherein the tag is selected from the group consisting of a HIS tail anda FLAG tail.
 25. The method of claim 23, wherein the tag is encoded by aPCR primer utilized in the step of PCR amplifying the allele encodingthe individual class I MHC molecule.
 26. The method of claim 24 whereinthe tag is encoded by the expression vector into which the PCR productis cloned.
 27. The method of claim 18, further comprising the step ofidentifying a source protein from which the at least one individual,endogenously loaded peptide ligand presented by the individual solubleclass I MHC molecule on the non-transfected cell line and not presentedby the individual soluble class I MHC molecule on the transfected cellline is obtained.
 28. The method of claim 18 wherein, in the step oftransfecting a portion of the non-transfected cell line, the portion ofthe non-transfected cell line is transfected with a gene from HIV. 29.The method of claim 18, wherein the transfected cell is further definedas a tumorigenic cell, and the non-transfected cell is further definedas a non-tumorigenic cell, whereby the step of transfecting a portion ofthe non-tumorigenic cell line with at least one of a gene from amicroorganism and a tumor gene, thereby provides a transformed,tumorigenic cell line.
 30. The method of claim 18, further comprisingthe step of: determining the source protein from which the at least oneendogenously loaded peptide ligand is obtained.
 31. The method of claim30, wherein the transfected cell is further defined as a tumorigeniccell, and the non-transfected cell is further defined as anon-tumorigenic cell, whereby the step of transfecting a portion of thenon-tumorigenic cell line with at least one of a gene from amicroorganism and a tumor gene, thereby provides a transformed,tumorigenic cell line.